Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Juong G.; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD; Li, Daqing

2007-01-01

Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into anmore » adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.« less

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

PubMed Central

Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

2015-01-01

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation

PubMed Central

Hrdličková, Radmila; Nehyba, Jiří; Liss, Andrew S.; Bose, Henry R.

2006-01-01

Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS. PMID:16352553

Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

PubMed Central

Edery, M; Rozakis-Adcock, M; Goujon, L; Finidori, J; Lévi-Meyrueis, C; Paly, J; Djiane, J; Postel-Vinay, M C; Kelly, P A

1993-01-01

A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients. Images PMID:8450064

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

PubMed

Wu, Liang; Zhang, Xiaolong; Zhao, Zhikun; Wang, Ling; Li, Bo; Li, Guibo; Dean, Michael; Yu, Qichao; Wang, Yanhui; Lin, Xinxin; Rao, Weijian; Mei, Zhanlong; Li, Yang; Jiang, Runze; Yang, Huan; Li, Fuqiang; Xie, Guoyun; Xu, Liqin; Wu, Kui; Zhang, Jie; Chen, Jianghao; Wang, Ting; Kristiansen, Karsten; Zhang, Xiuqing; Li, Yingrui; Yang, Huanming; Wang, Jian; Hou, Yong; Xu, Xun

2015-01-01

Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

Differential endothelial transcriptomics identifies semaphorin 3G as a vascular class 3 semaphorin.

PubMed

Kutschera, Simone; Weber, Holger; Weick, Anja; De Smet, Frederik; Genove, Guillem; Takemoto, Minoru; Prahst, Claudia; Riedel, Maria; Mikelis, Constantinos; Baulande, Sylvain; Champseix, Catherine; Kummerer, Petra; Conseiller, Emmanuel; Multon, Marie-Christine; Heroult, Melanie; Bicknell, Roy; Carmeliet, Peter; Betsholtz, Christer; Augustin, Hellmut G

2011-01-01

To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain-containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell-expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase.

PubMed

van Gennip, H G; van Rijn, P A; Widjojoatmodjo, M N; Moormann, R J

1999-03-01

A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.

Effects of CASP5 gene overexpression on angiogenesis of HMEC-1 cells.

PubMed

Li, Haiyan; Li, Yuzhen; Cai, Limin; Bai, Bingxue; Wang, Yanhua

2015-01-01

The efficacy of gene overexpression of CASP5, a caspase family member, in angiogenesis in vitro and its mechanisms were clarified. Human full-length CASP5 gene was delivered into human microvascular endothelial HMEC-1 cells by recombinant lentivirus. The infection was estimated by green fluorescent protein. MTT method was used to analyze the efficacy of gene overexpression in cell proliferation ability, and Matrigel was used to estimate its effects in angiogenesis ability of cells. Meanwhile, Western blot was used to analyze the effects of CASP5 gene overexpression on the expression levels of angpt-1, angpt-2, Tie2 and VEGF-1 in the cells, which were signaling pathway factors related to angiogenesis. Recombinant lentivirus containing human full-length CASP5 gene was packed and purified successfully, with virus titer of 1×10(8) TU/ml. The recombinant lentivirus was used to infect HMEC-1 cells with MOI of 1, leading to a cell infection rate of 100%. There were no significant effects of CASP5 gene overexpression on both cell proliferation ability and the expression level of angpt-1. Meanwhile, expressions of angpt-2 and VEGF-1 were both enhanced, while Tie2 expression was inhibited. Results indicated that CASP5 gene overexpression promoted angiogenesis of HMEC-1 cells. CASP5 gene overexpression significantly promoted angiogenesis ability of HMEC-1 cells, which was probably achieved by inhibiting angpt-1/Tie2 and promoting VEGF-1 signal pathway.

Hydroxyurea enhances SMN2 gene expression through nitric oxide release.

PubMed

Xu, Cheng; Chen, Xin; Grzeschik, Susanna M; Ganta, Madhuri; Wang, Ching H

2011-02-01

Small molecules that increase full-length survivor motor neuron (SMN) gene transcript are promising therapeutic candidates for spinal muscular atrophy (SMA). Hydroxyurea (HU) has recently been shown to increase full-length SMN transcript in cultured lymphocytes from patients with SMA. We investigate the mechanism by which HU enhances full-length SMN2 gene expression in SMA lymphocytes. Nitric oxide (NO) is a major intracellular metabolite of HU. We test whether NO donors can themselves enhance full-length SMN2 expression. Eighteen cell lines (five type I, five type II, six type III SMA, and two non-SMA controls) were treated with or without NO donors for 48 h. SMA cells treated with HU and three NO donors: two long-acting donors, Deta-NONOate and S-nitrosoglutathione, and one short-acting donor, 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, resulted in significant increase in full-length SMN2 mRNA. These effects were abolished by co-treatment with an NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide. One short-acting NO donor, S-nitroso-N-acetyl-DL-penicillamine, failed to show significant effect on full-length SMN2 expression, possibly due to high degree of cytotoxicity. These results were observed using both densitometry and quantitative PCR methods. We conclude that HU enhances SMN2 expression through the release of NO. NO donors may themselves be considered as new therapeutic candidates for SMA.

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

PubMed

Zhan, Jinghui; Felder, Barbara; Ellison, Aaron R; Winters, Aaron; Salimi-Moosavi, Hossein; Scully, Sheila; Turk, James R; Wei, Ping

2013-06-01

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

PubMed Central

Kutty, Geetha; England, Katherine J.; Kovacs, Joseph A.

2013-01-01

The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine–rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine–rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg. PMID:23532098

Altered regulation of ELAVL1/HuR in HLA-B27-expressing U937 monocytic cells.

PubMed

Sahlberg, Anna S; Ruuska, Marja; Granfors, Kaisa; Penttinen, Markus A

2013-01-01

To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response. U937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA-B27, or mutated HLA-B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA. Full length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27. Results show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5.

PubMed

Ward, T M; Iorns, E; Liu, X; Hoe, N; Kim, P; Singh, S; Dean, S; Jegg, A-M; Gallas, M; Rodriguez, C; Lippman, M; Landgraf, R; Pegram, M D

2013-05-09

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody-drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

Truncated p110 ERBB2 induces mammary epithelial cell migration, invasion and orthotopic xenograft formation, and is associated with loss of phosphorylated STAT5

PubMed Central

Ward, T M; Iorns, E; Liu, X; Hoe, N; Kim, P; Singh, S; Dean, S; Jegg, A-M; Gallas, M; Rodriguez, C; Lippman, M; Landgraf, R; Pegram, M D

2013-01-01

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5. PMID:22751112

Cloning and characterization of full length of a novel zebrafish gene Zsrg abundantly expressed in the germline stem cells.

PubMed

Lv, Daoyuan; Song, Ping; Chen, Yungui; Gong, Wuming; Mo, Saijun

2005-04-08

Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) (Accession No. BM316936), which came from zebrafish ovary and testis libraries. The full-length cDNA of this transcript was cloned and further confirmed by polymerase chain reaction and sequencing. The full-length cDNA of the novel gene is 807bp and encodes a novel protein of 187 amino acids, which shares no significant homology with any other known proteins. Characterization of genomic sequences of the gene revealed that it spans 6kb on the linkage group 3 and is composed of five exons and four introns. RT-PCR analysis showed that it was expressed in mature oocytes and one-cell stage, and persisted until 24h of development. RT-PCR also revealed that it is expressed in gonad and kidney, with the highest level of expression in the testis. The expression sites of the novel gene in adult gonad were further localized by in situ hybridization to oogonia and growing oocytes in ovary and to spermatogonia, spermatocytes but not to spermatids in testis. Based on its abundance in testis and the germline stem cell-spermatogonia and oogonia, we hypothesize that it may function as a testicular development and gametogenesis related gene that plays important roles in spermatogenesis, and named it Zsrg (zebrafish testis spermatogenesis related gene, Zsrg).

Decreased expression of MUC1 induces apoptosis and inhibits migration in pancreatic cancer PANC-1 cells via regulation of Slug pathway.

PubMed

Zhao, Ping; Meng, Meng; Xu, Bin; Dong, Aiping; Ni, Guangzhen; Lu, Lianfang

2017-12-06

MUC1, a membrane tethered mucin glycoprotein, is overexpressed in > 60% of human pancreatic cancers (PCs), and is associated with poor prognosis and enhanced metastasis. Here, we report the effect of silencing MUC1 expression on the growth, migration and invasive ability of pancreatic cancer cells, and explored its mechanisms. We observed that siRNA mediated suppression of the MUC1 expression significantly reduced invasive and migrative capability and induced apoptosis of the pancreatic cancer PANC-1 cells. We found that Slug was inhibited in the MUC1 siRNA transfected PANC-1 cells (MUC1 siRNA/PANC-1 cells). Expression of PUMA and E-cadherin was increased in the MUC1 siRNA/PANC-1 cells. PANC-1 cells overexpressing full long Slug gene (when transfected with Slug cDNA plasmid) significantly inhibited PUMA and E-cadherin expression in the MUC1 siRNA/PANC-1 cells. Silencing PUMA expression inhibited apoptosis in the MUC1 siRNA transfected PANC-1 cells (MUC1 siRNA/PANC-1 cells). Silencing E-cadherin expression restored the invasion and migration ability in the MUC1 siRNA/PANC-1 cells. We therefore concluded that silencing MUC1 expression inhibited migration and invasion, and induced apoptosis of PANC-1 cells via downregulation of Slug and upregulation of Slug dependent PUMA and E-cadherin expression. MUC1 could serve as a potential therapeutic target in pancreatic cancer.

Characterization of Toll-like receptor 3 gene in large yellow croaker, Pseudosciaena crocea.

PubMed

Huang, Xue-Na; Wang, Zhi-Yong; Yao, Cui-Luan

2011-07-01

Toll-like receptor 3 (TLR3) plays an important role in innate immune responses. In this report, the full-length cDNA sequence and genomic structure of Pseudosciaena crocea TLR3 (PcTLR3) were identified and characterized. The full-length cDNA of PcTLR3 was of 3384 bp, including a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 589 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The full-length genome sequence of PcTLR3 was composed of 5721 nucleotides, including five exons and four introns. The putative PcTLR3 protein contained a signal peptide sequence, 16 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR3 in most tissues, with the predominant expression in liver, then intestine, and the weakest expression in blood cells. The expression of PcTLR3 after injection with poly inosinic:cytidylic (I:C) and Vibrio parahemolyticus was tested in spleen, blood cells and liver. The results indicated that PcTLR3 transcripts could be induced in the three tissues by injection with poly I:C. The highest expression was in the blood cells with 43.5 times (at 6h) greater expression than in the control (p<0.05). In addition, after V. parahemolyticus challenge, a moderate up-regulation and down-regulation of PcTLR3 was found in blood cells and liver, respectively. Our results suggested that PcTLR3 might play an important role in fish's defense against both viral and bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.

PubMed

Aagaard, Lars; Mikkelsen, Jacob Giehm; Warming, Søren; Duch, Mogens; Pedersen, Finn Skou

2002-02-01

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

Pharmacological characterization of extracellular acidification rate responses in human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells

PubMed Central

Coldwell, M C; Boyfield, I; Brown, A M; Stemp, G; Middlemiss, D N

1999-01-01

This study characterized pharmacologically the functional responses to agonists at human dopamine D2(long) (hD2), D3 (hD3) and D4.4 (hD4) zreceptors separately expressed in cloned cells using the cytosensor microphysiometer. Dopaminergic receptor agonists caused increases in extracellular acidification rate in adherent Chinese hamster ovary (CHO) clones expressing hD2, hD3 or hD4 receptors. Acidification rate responses to agonists in other cell lines expressing these receptors were smaller than those in adherent CHO cells. The time courses and maximum increases in acidification rate of the agonist responses in adherent CHO cells were different between the three dopamine receptor clones. Responses were blocked by pretreatment of cells with pertussis toxin or amiloride analogues. Most agonists had full intrinsic activity at each of the dopamine receptor subtypes, as compared to quinpirole, however both enantiomers of UH-232 and (−)3-PPP were partial agonists in this assay system. The functional potency of full agonists at each of the three receptors expressed in CHO cells was either higher than, or similar to, the apparent inhibition constants (Ki) determined in [125I]-iodosulpride competition binding studies. Functional selectivities of the agonists were less than radioligand binding selectivities. The rank orders of agonist potencies and selectivities were similar, but not identical, to the rank orders of radioligand binding affinities and selectivities. The dopamine receptor antagonists, iodosulpride and clozapine, had no effect on basal acidification rates but inhibited acidification responses in CHO cells to quinpirole in an apparently competitive manner. Antagonist potencies closely matched their radioligand binding affinities in these cells. PMID:10455259

Alpha-latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in beta-cells.

PubMed

Lajus, Sophie; Vacher, Pierre; Huber, Denise; Dubois, Mathilde; Benassy, Marie-Noëlle; Ushkaryov, Yuri; Lang, Jochen

2006-03-03

The spider venom alpha-latrotoxin (alpha-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, alpha-LTX first induced membrane depolarization by inhibition of repolarizing K(+) channels followed by the appearance of Ca(2+) transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca(2+)](i)) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX(N4C), which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K(+) channels via phospholipase C, activated L-type Ca(2+) channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca(2+)](i) in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca(2+) channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, alpha-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K(+) and Ca(2+) channels as novel targets of its secretory activity.

Expression of connective tissue growth factor (CTGF/CCN2) in breast cancer cells is associated with increased migration and angiogenesis.

PubMed

Chien, Wenwen; O'Kelly, James; Lu, Daning; Leiter, Amanda; Sohn, Julia; Yin, Dong; Karlan, Beth; Vadgama, Jay; Lyons, Karen M; Koeffler, H Phillip

2011-06-01

Connective tissue growth factor (CTGF/CCN2) belongs to the CCN family of matricellular proteins, comprising Cyr61, CTGF, NovH and WISP1-3. The CCN proteins contain an N-terminal signal peptide followed by four conserved domains sharing sequence similarities with the insulin-like growth factor binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a C-terminal growth factor cysteine knot domain. To investigate the role of CCN2 in breast cancer, we transfected MCF-7 cells with full-length CCN2, and with four mutant constructs in which one of the domains had been deleted. MCF-7 cells stably expressing full-length CCN2 demonstrated reduced cell proliferation, increased migration in Boyden chamber assays and promoted angiogenesis in chorioallantoic membrane assays compared to control cells. Deletion of the C-terminal cysteine knot domain, but not of any other domain-deleted mutants, abolished activities mediated by full-length CCN2. We have dissected the role of CCN2 in breast tumorigenesis on a structural basis.

Parallels between Global Transcriptional Programs of Polarizing Caco-2 Intestinal Epithelial Cells In Vitro and Gene Expression Programs in Normal Colon and Colon Cancer

PubMed Central

Sääf, Annika M.; Halbleib, Jennifer M.; Chen, Xin; Yuen, Siu Tsan; Leung, Suet Yi

2007-01-01

Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell–cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2. PMID:17699589

Novel cell-cell signaling by microglial transmembrane TNFα with implications for neuropathic pain

PubMed Central

Zhou, Zhigang; Peng, Xiangmin; Hagshenas, Jafar; Insolera, Ryan; Fink, David J.; Mata, Marina

2010-01-01

Neuropathic pain is accompanied by neuroimmune activation in dorsal horn of spinal cord. We have observed that in animal models this activation is characterized by increased expression of transmembrane tumor necrosis factor α (mTNFα) without release of soluble (sTNFα). Here we report that the pain-related neurotransmitter peptide substance P (SP) increases expression of mTNFα without release of sTNFα from primary microglial cells. We modeled this interaction using an immortalized microglial cell line; exposure of these cells to SP also resulted in increased expression of mTNFα but without any increase in expression of the TNF-cleaving enzyme (TACE) and no release of sTNFα. In order to evaluate the biological function of uncleaved mTNFα, we transfected COS-7 cells with a mutant full length TNFα construct resistant to cleavage by TACE. Co-culture of COS-7 cells expressing the mutant TNFα with microglial cells led to microglial cell activation indicated by increased OX-42 immunoreactivity and release of macrophage chemoattractant peptide 1 (CCL2) by direct cell-cell contact. These results suggest a novel pathway through which release of SP by primary afferents activates microglial expression of mTNFα, establishing a feed-forward loop that may contribute to the establishment of chronic pain. PMID:20609516

A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

PubMed Central

Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

2014-01-01

Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function.

PubMed

Basit, Abdul; Tang, Wenwen; Wu, Dianqing

2016-01-01

To silence genes in neutrophils efficiently, we exploited the RNA interference and developed an shRNA-based gene knockdown technique. This method involves transfection of mouse bone marrow-derived hematopoietic stem cells with retroviral vector carrying shRNA directed at a specific gene. Transfected stem cells are then transplanted into irradiated wild-type mice. After engraftment of stem cells, the transplanted mice have two sets of circulating neutrophils. One set has a gene of interest knocked down while the other set has full complement of expressed genes. This efficient technique provides a unique way to directly compare the response of neutrophils with a knocked-down gene to that of neutrophils with the full complement of expressed genes in the same environment.

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells.

PubMed

Hsieh, Chen-Lin; Cai, Changmeng; Giwa, Ahmed; Bivins, Aaronica; Chen, Shao-Yong; Sabry, Dina; Govardhan, Kumara; Shemshedini, Lirim

2008-07-01

Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.

Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

PubMed

Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

2009-01-20

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

PubMed

Locker, Morgane; Kellermann, Odile; Boucquey, Marie; Khun, Huot; Huerre, Michel; Poliard, Anne

2004-01-01

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

A novel apoptosis correlated molecule: expression and characterization of protein Latcripin-1 from Lentinula edodes C(91-3).

PubMed

Liu, Ben; Zhong, Mintao; Lun, Yongzhi; Wang, Xiaoli; Sun, Wenchang; Li, Xingyun; Ning, Anhong; Cao, Jing; Zhang, Wei; Liu, Lei; Huang, Min

2012-01-01

An apoptosis correlated molecule-protein Latcripin-1 of Lentinula edodes C(91-3)-was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C(91-3). According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3'-Full Rapid Amplification of cDNA Ends (RACE) and 5'-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins.

A Novel Apoptosis Correlated Molecule: Expression and Characterization of Protein Latcripin-1 from Lentinula edodes C91–3

PubMed Central

Liu, Ben; Zhong, Mintao; Lun, Yongzhi; Wang, Xiaoli; Sun, Wenchang; Li, Xingyun; Ning, Anhong; Cao, Jing; Zhang, Wei; Liu, Lei; Huang, Min

2012-01-01

An apoptosis correlated molecule—protein Latcripin-1 of Lentinula edodes C91–3—was expressed and characterized in Pichia pastoris GS115. The total RNA was obtained from Lentinula edodes C91–3. According to the transcriptome, the full-length gene of Latcripin-1 was isolated with 3′-Full Rapid Amplification of cDNA Ends (RACE) and 5′-Full RACE methods. The full-length gene was inserted into the secretory expression vector pPIC9K. The protein Latcripin-1 was expressed in Pichia pastoris GS115 and analyzed by Sodium Dodecylsulfonate Polyacrylate Gel Electrophoresis (SDS-PAGE) and Western blot. The Western blot showed that the protein was expressed successfully. The biological function of protein Latcripin-1 on A549 cells was studied with flow cytometry and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) method. The toxic effect of protein Latcripin-1 was detected with the MTT method by co-culturing the characterized protein with chick embryo fibroblasts. The MTT assay results showed that there was a great difference between protein Latcripin-1 groups and the control group (p < 0.05). There was no toxic effect of the characterized protein on chick embryo fibroblasts. The flow cytometry showed that there was a significant difference between the protein groups of interest and the control group according to apoptosis function (p < 0.05). At the same time, cell ultrastructure observed by transmission electron microscopy supported the results of flow cytometry. The work demonstrates that protein Latcripin-1 can induce apoptosis of human lung cancer cells A549 and brings new insights into and advantages to finding anti-tumor proteins. PMID:22754362

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chetty, Chandramu; Dontula, Ranadheer; Ganji, Purnachandra Nagaraju

2012-01-13

Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reductionmore » in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.« less

Altered Regulation of ELAVL1/HuR in HLA-B27–Expressing U937 Monocytic Cells

PubMed Central

Sahlberg, Anna S.; Ruuska, Marja; Granfors, Kaisa; Penttinen, Markus A.

2013-01-01

Objective To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response. Methods U937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA–B27, or mutated HLA–B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA. Results Full length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27. Conclusion Results show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response. PMID:23894643

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule.

PubMed

Yang, Muhua; Adla, Shalini; Temburni, Murali K; Patel, Vivek P; Lagow, Errin L; Brady, Owen A; Tian, Jing; Boulos, Magdy I; Galileo, Deni S

2009-10-29

Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding alphavbeta5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain.

Stimulation of glioma cell motility by expression, proteolysis, and release of the L1 neural cell recognition molecule

PubMed Central

Yang, Muhua; Adla, Shalini; Temburni, Murali K; Patel, Vivek P; Lagow, Errin L; Brady, Owen A; Tian, Jing; Boulos, Magdy I; Galileo, Deni S

2009-01-01

Background Malignant glioma cells are particularly motile and can travel diffusely through the brain parenchyma, apparently without following anatomical structures to guide their migration. The neural adhesion/recognition protein L1 (L1CAM; CD171) has been implicated in contributing to stimulation of motility and metastasis of several non-neural cancer types. We explored the expression and function of L1 protein as a stimulator of glioma cell motility using human high-grade glioma surgical specimens and established rat and human glioma cell lines. Results L1 protein expression was found in 17 out of 18 human high-grade glioma surgical specimens by western blotting. L1 mRNA was found to be present in human U-87/LacZ and rat C6 and 9L glioma cell lines. The glioma cell lines were negative for surface full length L1 by flow cytometry and high resolution immunocytochemistry of live cells. However, fixed and permeablized cells exhibited positive staining as numerous intracellular puncta. Western blots of cell line extracts revealed L1 proteolysis into a large soluble ectodomain (~180 kDa) and a smaller transmembrane proteolytic fragment (~32 kDa). Exosomal vesicles released by the glioma cell lines were purified and contained both full-length L1 and the proteolyzed transmembrane fragment. Glioma cell lines expressed L1-binding αvβ5 integrin cell surface receptors. Quantitative time-lapse analyses showed that motility was reduced significantly in glioma cell lines by 1) infection with an antisense-L1 retroviral vector and 2) L1 ectodomain-binding antibodies. Conclusion Our novel results support a model of autocrine/paracrine stimulation of cell motility in glioma cells by a cleaved L1 ectodomain and/or released exosomal vesicles containing L1. This mechanism could explain the diffuse migratory behavior of high-grade glioma cancer cells within the brain. PMID:19874583

Epidermal Growth Factor-Dependent Transformation by a Human EGF Receptor Proto-Oncogene

NASA Astrophysics Data System (ADS)

Velu, Thierry J.; Beguinot, Laura; Vass, William C.; Willingham, Mark C.; Merlino, Glenn T.; Pastan, Ira; Lowy, Douglas R.

1987-12-01

The epidermal growth factor (EGF) receptor gene EGFR has been placed in a retrovirus vector to examine the growth properties of cells that experimentally overproduce a full-length EGF receptor. NIH 3T3 cells transfected with the viral DNA or infected with the corresponding rescued retrovirus developed a fully transformed phenotype in vitro that required both functional EGFR expression and the presence of EGF in the growth medium. Cells expressing 4 × 105 EGF receptors formed tumors in nude mice, while control cells did not. Therefore, the EGFR retrovirus, which had a titer on NIH 3T3 cells that was greater than 107 focus-forming units per milliliter, can efficiently transfer and express this gene, and increased numbers of EGF receptors can contribute to the transformed phenotype.

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

PubMed Central

Kortmann, Jens; Brubaker, Sky W.

2015-01-01

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

PubMed

van Rijn, Jorik M; Chandra Ardy, Rico; Kuloğlu, Zarife; Härter, Bettina; van Haaften-Visser, Désirée Y; van der Doef, Hubert P J; van Hoesel, Marliek; Kansu, Aydan; van Vugt, Anke H M; Ng, Marini; Kokke, Freddy T M; Krolo, Ana; Başaran, Meryem Keçeli; Kaya, Neslihan Gurcan; Aksu, Aysel Ünlüsoy; Dalgıç, Buket; Ozcay, Figen; Baris, Zeren; Kain, Renate; Stigter, Edwin C A; Lichtenbelt, Klaske D; Massink, Maarten P G; Duran, Karen J; Verheij, Joke B G M; Lugtenberg, Dorien; Nikkels, Peter G J; Brouwer, Henricus G F; Verkade, Henkjan J; Scheenstra, Rene; Spee, Bart; Nieuwenhuis, Edward E S; Coffer, Paul J; Janecke, Andreas R; van Haaften, Gijs; Houwen, Roderick H J; Müller, Thomas; Middendorp, Sabine; Boztug, Kaan

2018-03-29

Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequence analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for activities of caspases 3 and 7. In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Walleye dermal sarcoma virus: expression of a full-length clone or the rv-cyclin (orf a) gene is cytopathic to the host and human tumor cells.

PubMed

Xu, Kun; Zhang, Ting Ting; Wang, Ling; Zhang, Cun Fang; Zhang, Long; Ma, Li Xia; Xin, Ying; Ren, Chong Hua; Zhang, Zhi Qiang; Yan, Qiang; Martineau, Daniel; Zhang, Zhi Ying

2013-02-01

Walleye dermal sarcoma virus (WDSV) is etiologically associated with a skin tumor, walleye dermal sarcoma (WDS), which develops in the fall and regresses in the spring. WDSV genome contains, in addition to gag, pol and env, three open reading frames (orfs) designated orf a (rv-cyclin), orf b and orf c. Unintegrated linear WDSV provirus DNA isolated from infected tumor cells was used to construct a full-length WDSV provirus clone pWDSV, while orf a was cloned into pSVK3 to construct the expression vector porfA. Stable co-transfection of a walleye cell line (W12) with pWDSV and pcDNA3 generated fewer and smaller G418-resistant colonies compared to the control. By Northern blot analysis, several small transcripts (2.8, 1.8, 1.2, and 0.8 kb) were detected using a WDSV LTR-specific probe. By RT-PCR and Southern blot analysis, three cDNAs (2.4, 1.6 and 0.8 kb) were identified, including both orf a and orf b messenger. Furthermore stable co-transfection of both a human lung adenocarcinoma cell line (SPC-A-1) and a cervical cancer cell line (HeLa) with pcDNA3 and ether porfA or pWDSV also generated fewer and smaller G418-resistant colonies. We conclude that expression of the full-length WDSV clone or the orf a gene inhibits the host fish and human tumor cell growth, and Orf A protein maybe a potential factor which contributes to the seasonal tumor development and regression. This is the first fish provirus clone that has been expressed in cell culture system, which will provide a new in vitro model for tumor research and oncotherapy study.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

PubMed

Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

2014-06-24

CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

PubMed Central

Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G.; Nghiem, Paul

2013-01-01

Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region. PMID:23514892

[Construction of the superantigen SEA transfected laryngocarcinoma cells].

PubMed

Ji, Xiaobin; Jingli, J V; Liu, Qicai; Xie, Jinghua

2013-04-01

To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients

PubMed Central

Bagdonaite, Ieva; Wandall, Hans H.; Litvinov, Ivan V.; Nastasi, Claudia; Becker, Jürgen C.; Dabelsteen, Sally; Geisler, Carsten; Bonefeld, Charlotte M.; Zhang, Qian; Wasik, Mariusz A.; Zhou, Youwen; Sasseville, Denis; Ødum, Niels; Woetmann, Anders

2015-01-01

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22ΔN), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22ΔN mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22ΔN (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22ΔN and CD22wt in these cells. In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients. PMID:25957418

Ectopic expression of a novel CD22 splice-variant regulates survival and proliferation in malignant T cells from cutaneous T cell lymphoma (CTCL) patients.

PubMed

Bagdonaite, Ieva; Wandall, Hans H; Litvinov, Ivan V; Nastasi, Claudia; Becker, Jürgen C; Dabelsteen, Sally; Geisler, Carsten; Bonefeld, Charlotte M; Zhang, Qian; Wasik, Mariusz A; Zhou, Youwen; Sasseville, Denis; Ødum, Niels; Woetmann, Anders

2015-06-10

CD22 is a member of the Sialic acid-binding Ig-like lectin (Siglec) family of lectins described to be exclusively present in B lymphocytes and B cell-derived neoplasms. Here, we describe a novel splice form of CD22 (designated CD22∆N), which lacks the N-terminal domain as demonstrated by exon-specific RT-PCR and differential recognition by anti-CD22 antibodies. Importantly, CD22∆N mRNA is expressed in skin lesions from 39 out of 60 patients with cutaneous T cell lymphoma (CTCL), whereas few patients (6 out of 60) expresses full-length, wild type CD22 (CD22wt). In addition, IHC staining of tumor biopsies confirmed the expression of CD22 in CD4+ T cells. Moreover, four out of four malignant T cell lines express CD22: Two cell lines express CD22∆N (MyLa2059 and PB2B) and two express CD22wt (MAC-1 and MAC-2A). siRNA-mediated silencing of CD22 impairs proliferation and survival of malignant T cells, demonstrating a functional role for both CD22∆N and CD22wt in these cells.In conclusion, we provide the first evidence for an ectopic expression of CD22 and a novel splice variant regulating malignant proliferation and survival in CTCL. Analysis of expression and function of CD22 in cutaneous lymphomas may form the basis for development of novel targeted therapies for our patients.

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line.

PubMed

Flood, Fiona; Sundström, Erik; Samuelsson, Eva-Britt; Wiehager, Birgitta; Seiger, Ake; Johnston, Janet A; Cowburn, Richard F

2004-06-01

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.

Lactobacillus bulgaricus Proteinase Expressed in Lactococcus lactis Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins

PubMed Central

Bernasconi, Eric; Germond, Jacques-Edouard; Delley, Michèle; Fritsché, Rodolphe; Corthésy, Blaise

2002-01-01

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB∂-BLG yielded up to 170 μg per 109 CFU in the culture supernatant and 9 μg per 109 CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. PMID:12039750

Cancer induction by restriction of oncogene expression to the stem cell compartment

PubMed Central

Pérez-Caro, María; Cobaleda, César; González-Herrero, Inés; Vicente-Dueñas, Carolina; Bermejo-Rodríguez, Camino; Sánchez-Beato, Margarita; Orfao, Alberto; Pintado, Belén; Flores, Teresa; Sánchez-Martín, Manuel; Jiménez, Rafael; Piris, Miguel A; Sánchez-García, Isidro

2009-01-01

In human cancers, all cancerous cells carry the oncogenic genetic lesions. However, to elucidate whether cancer is a stem cell-driven tissue, we have developed a strategy to limit oncogene expression to the stem cell compartment in a transgenic mouse setting. Here, we focus on the effects of the BCR-ABLp210 oncogene, associated with chronic myeloid leukaemia (CML) in humans. We show that CML phenotype and biology can be established in mice by restricting BCR-ABLp210 expression to stem cell antigen 1 (Sca1)+ cells. The course of the disease in Sca1-BCR-ABLp210 mice was not modified on STI571 treatment. However, BCR-ABLp210-induced CML is reversible through the unique elimination of the cancer stem cells (CSCs). Overall, our data show that oncogene expression in Sca1+ cells is all that is required to fully reprogramme it, giving rise to a full-blown, oncogene-specified tumour with all its mature cellular diversity, and that elimination of the CSCs is enough to eradicate the whole tumour. PMID:19037256

NK cell-released exosomes: Natural nanobullets against tumors.

PubMed

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

NK cell-released exosomes

PubMed Central

Fais, Stefano

2013-01-01

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. PMID:23482694

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

PubMed

Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

2014-03-01

To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

PubMed

Wang, Kening; Kappel, Justin D; Canders, Caleb; Davila, Wilmer F; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I

2012-12-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

PubMed Central

Kappel, Justin D.; Canders, Caleb; Davila, Wilmer F.; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I.

2012-01-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine. PMID:22993162

Research Resource: Global Identification of Estrogen Receptor β Target Genes in Triple Negative Breast Cancer Cells

PubMed Central

Shanle, Erin K.; Zhao, Zibo; Hawse, John; Wisinski, Kari; Keles, Sunduz; Yuan, Ming

2013-01-01

Breast cancers that are negative for estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 are known as triple-negative breast cancers (TNBC). TNBCs are associated with an overall poor prognosis because they lack expression of therapeutic targets like ERα and are biologically more aggressive. A second estrogen receptor, ERβ, has been found to be expressed in 50% to 90% of ERα-negative breast cancers, and ERβ expression in TNBCs has been shown to correlate with improved disease-free survival and good prognosis. To elucidate the role of ERβ in regulating gene expression and cell proliferation in TNBC cells, the TNBC cell line MDA-MB-468 was engineered with inducible expression of full-length ERβ. In culture, ERβ expression inhibited cell growth by inducing a G1 cell cycle arrest, which was further enhanced by 17β-estradiol treatment. In xenografts, ERβ expression also inhibited tumor formation and growth, and 17β-estradiol treatment resulted in rapid tumor regression. Furthermore, genomic RNA sequencing identified both ligand-dependent and -independent ERβ target genes, some of which were also regulated by ERβ in other TNBC cell lines and correlated with ERβ expression in a cohort of TNBCs from the Cancer Genome Atlas Network. ERβ target genes were enriched in genes that regulate cell death and survival, cell movement, cell development, and growth and proliferation, as well as genes involved in the Wnt/β-catenin and the G1/S cell cycle phase checkpoint pathways. In addition to confirming the anti-proliferative effects of ERβ in TNBC cells, these data provide a comprehensive resource of ERβ target genes and suggest that ERβ may be targeted with ligands that can stimulate its growth inhibitory effects. PMID:23979844

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

Clinical and molecular implications of mosaicism in FMR1 full mutations

PubMed Central

Pretto, Dalyir; Yrigollen, Carolyn M.; Tang, Hiu-Tung; Williamson, John; Espinal, Glenda; Iwahashi, Chris K.; Durbin-Johnson, Blythe; Hagerman, Randi J.; Hagerman, Paul J.; Tassone, Flora

2014-01-01

Expansions of more than 200 CGG repeats (full mutation) in the FMR1 gene give rise to fragile X syndrome (FXS) through a process that generally involves hypermethylation of the FMR1 promoter region and gene silencing, resulting in absence of expression of the encoded protein, FMRP. However, mosaicism with alleles differing in size and extent of methylation often exist within or between tissues of individuals with FXS. In the current work, CGG-repeat lengths and methylation status were assessed for eighteen individuals with FXS, including 13 mosaics, for which peripheral blood cells (PBMCs) and primary fibroblast cells were available. Our results show that for both PBMCs and fibroblasts, FMR1 mRNA and FMRP expression are directly correlated with the percent of methylation of the FMR1 allele. In addition, Full Scale IQ scores were inversely correlated with the percent methylation and positively correlated with higher FMRP expression. These latter results point toward a positive impact on cognition for full mutation mosaics with lower methylation compared to individuals with fully methylated, full mutation alleles. However, we did not observe a significant reduction in the number of seizures, nor in the severity of hyperactivity or autism spectrum disorder, among individuals with mosaic genotypes in the presentation of FXS. These observations suggest that low, but non-zero expression of FMRP may be sufficient to positively impact cognitive function in individuals with FXS, with methylation mosaicism (lowered methylation fraction) contributing to a more positive clinical outcome. PMID:25278957

Identification of apoptosis-related PLZF target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

2007-07-27

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

PubMed Central

Blanc, Andrea Maria; Berois, Mabel Beatriz; Tomé, Lorena Magalí; Epstein, Alberto L.

2012-01-01

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease. PMID:22437537

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

PubMed Central

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

2013-01-01

Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

PubMed Central

Ning, Jin-Ying; Sun, Guo-Xun; Huang, Su; Ma, Hong; An, Ping; Meng, Lin; Song, Shu-Mei; Wu, Jian; Shou, Cheng-Chao

2003-01-01

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation. PMID:14562370

C/EBPβ LIP augments cell death by inducing osteoglycin.

PubMed

Wassermann-Dozorets, Rina; Rubinstein, Menachem

2017-04-06

Many types of tumor cell are devoid of the extracellular matrix proteoglycan osteoglycin (Ogn), but its role in tumor biology is poorly studied. Here we show that RNAi of Ogn attenuates stress-triggered cell death, whereas its overexpression increases cell death. We found that the transcription factor C/EBPβ regulates the expression of Ogn. C/EBPβ is expressed as a full-length, active form (LAP) and as a truncated, dominant-negative form (LIP), and the LIP/LAP ratio is positively correlated with the extent of cell death under stress. For example, we reported that drug-resistant tumor cells lack LIP altogether, and its supplementation abolished their resistance to chemotherapy and to endoplasmic reticulum (ER) stress. Here we further show that elevated LIP/LAP ratio robustly increased Ogn expression and cell death under stress by modulating the mitogen-activated protein kinase/activator protein 1 pathway (MAPK/AP-1). Our findings suggest that LIP deficiency renders tumor cell resistant to ER stress by preventing the induction of Ogn.

Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

PubMed

Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

2013-01-01

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133– metastatic colon cancer cells initiate tumors

PubMed Central

Shmelkov, Sergey V.; Butler, Jason M.; Hooper, Andrea T.; Hormigo, Adilia; Kushner, Jared; Milde, Till; St. Clair, Ryan; Baljevic, Muhamed; White, Ian; Jin, David K.; Chadburn, Amy; Murphy, Andrew J.; Valenzuela, David M.; Gale, Nicholas W.; Thurston, Gavin; Yancopoulos, George D.; D’Angelica, Michael; Kemeny, Nancy; Lyden, David; Rafii, Shahin

2008-01-01

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10–/–CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133– population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133– metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133– cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24–), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133– subset, which is also capable of tumor initiation in NOD/SCID mice. PMID:18497886

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture.

PubMed

Wang, Jun; Ti, Yunfan; Wang, Yicun; Guo, Guodong; Jiang, Hui; Chang, Menghan; Qian, Hongbo; Zhao, Jianning; Sun, Guojing

2018-04-19

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4 + T cells in patients with long bone fracture. We found that LAG-3 + cells represented approximately 13% of peripheral blood CD4 + T cells on average. Compared to LAG-3 - CD4 + T cells, LAG-3 + CD4 + T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3 + CD4 + T cells expressed significantly higher levels of IL-10 and TGF-β than LAG-3 - CD4 + T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3 + CD4 + T cells. The frequency of LAG-3 + CD4 + T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4 + T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1

PubMed Central

YANG, LIANG; LIU, REN; MA, HONG-BIN; YING, MING-ZHEN; WANG, YA-JIE

2015-01-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest. PMID:26622693

Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1.

PubMed

Yang, Liang; Liu, Ren; Ma, Hong-Bin; Ying, Ming-Zhen; Wang, Ya-Jie

2015-09-01

The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 ( GSTP1 ) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G 2 /M phase arrest in the GSTP1 -expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1 -expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G 2 /M phase arrest.

Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening.

PubMed

Dansithong, Warunee; Paul, Sharan; Scoles, Daniel R; Pulst, Stefan M; Huynh, Duong P

2015-01-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z'-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA promoter and 5'-UTR.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

PubMed

Kirilyuk, Alexander; Tolstonog, Genrich V; Damert, Annette; Held, Ulrike; Hahn, Silvia; Löwer, Roswitha; Buschmann, Christian; Horn, Axel V; Traub, Peter; Schumann, Gerald G

2008-02-01

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Statistical indicators of collective behavior and functional clusters in gene networks of yeast

NASA Astrophysics Data System (ADS)

Živković, J.; Tadić, B.; Wick, N.; Thurner, S.

2006-03-01

We analyze gene expression time-series data of yeast (S. cerevisiae) measured along two full cell-cycles. We quantify these data by using q-exponentials, gene expression ranking and a temporal mean-variance analysis. We construct gene interaction networks based on correlation coefficients and study the formation of the corresponding giant components and minimum spanning trees. By coloring genes according to their cell function we find functional clusters in the correlation networks and functional branches in the associated trees. Our results suggest that a percolation point of functional clusters can be identified on these gene expression correlation networks.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton's jelly stem cells (hWJSCs).

PubMed

Mostafavi-Pour, Zohreh; Ashrafi, Mohammad Reza; Talaei-Khozani, Tahereh

2018-06-01

Human Wharton's jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P < 0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.

The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

PubMed

Bellantuono, I; Lashford, L S; Rafferty, J A; Fairbairn, L J

2000-05-01

As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

Development and characterization of a eukaryotic expression system for human type II procollagen.

PubMed

Wieczorek, Andrew; Rezaei, Naghmeh; Chan, Clara K; Xu, Chuan; Panwar, Preety; Brömme, Dieter; Merschrod S, Erika F; Forde, Nancy R

2015-12-15

Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

PubMed

Obata, Yuuki; Kimura, Shunsuke; Nakato, Gaku; Iizuka, Keito; Miyagawa, Yurika; Nakamura, Yutaka; Furusawa, Yukihiro; Sugiyama, Machiko; Suzuki, Keiichiro; Ebisawa, Masashi; Fujimura, Yumiko; Yoshida, Hisahiro; Iwanaga, Toshihiko; Hase, Koji; Ohno, Hiroshi

2014-12-01

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj-a gene encoding a transcription factor implicated in Notch signaling-in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE. © 2014 The Authors.

Downregulation of missing in metastasis gene (MIM) is associated with the progression of bladder transitional carcinomas.

PubMed

Wang, Ying; Liu, Jiali; Smith, Elizabeth; Zhou, Kang; Liao, Jie; Yang, Guang-Yu; Tan, Ming; Zhan, Xi

2007-03-01

Missing in metastasis (MIM) gene encodes a putative metastasis suppressor. However, the role of MIM in tumorigenesis and metastasis has not yet been established. Western blot analysis using a MIM specific antibody demonstrated that MIM protein is present at varying levels in a variety of normal cells as well as tumor cell lines. Immunohistochemical staining of adult mouse tissues revealed abundant MIM immunoreactivity in uroepithelial cells in the bladder, neuron Purkinje cells in the cerebellum, and megakaryocytes in the bone marrow and spleen in addition. MIM immunoreactivity also was found in human normal bladder transitional epithelial cells. However, the reactivity was not seen in 69 percent of human primary transitional cell carcinoma specimens. Over 51 percent of the tumors at low grade display MIM staining similarly to the normal cells, whereas only 16.7 percent of the tumors at high-grade with poor differentiation show faint or mild staining. Furthermore, full-length MIM protein is highly expressed in SV-HUC-L an immortalized normal transitional epithelial cell line, moderately expressed in T24 and poorly expressed in J82 and TCCSUP transitional cell carcinoma cells. This finding indicates that downegulation of MIM expression may correlate with the transition of tumor cells from distinct epithelium-like morphology to less differentiated carcinomas.

Activated Leukocyte Cell Adhesion Molecule Expression and Shedding in Thyroid Tumors

PubMed Central

Miccichè, Francesca; Da Riva, Luca; Fabbi, Marina; Pilotti, Silvana; Mondellini, Piera; Ferrini, Silvano; Canevari, Silvana; Pierotti, Marco A.; Bongarzone, Italia

2011-01-01

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is expressed in various tissues, cancers, and cancer-initiating cells. Alterations in expression of ALCAM have been reported in several human tumors, and cell adhesion functions have been proposed to explain its association with cancer. Here we documented high levels of ALCAM expression in human thyroid tumors and cell lines. Through proteomic characterization of ALCAM expression in the human papillary thyroid carcinoma cell line TPC-1, we identified the presence of a full-length membrane-associated isoform in cell lysate and of soluble ALCAM isoforms in conditioned medium. This finding is consistent with proteolytically shed ALCAM ectodomains. Nonspecific agents, such as phorbol myristate acetate (PMA) or ionomycin, provoked increased ectodomain shedding. Epidermal growth factor receptor stimulation also enhanced ALCAM secretion through an ADAM17/TACE-dependent pathway. ADAM17/TACE was expressed in the TPC-1 cell line, and ADAM17/TACE silencing by specific small interfering RNAs reduced ALCAM shedding. In addition, the CGS27023A inhibitor of ADAM17/TACE function reduced ALCAM release in a dose-dependent manner and inhibited cell migration in a wound-healing assay. We also provide evidence for the existence of novel O-glycosylated forms and of a novel 60-kDa soluble form of ALCAM, which is particularly abundant following cell stimulation by PMA. ALCAM expression in papillary and medullary thyroid cancer specimens and in the surrounding non-tumoral component was studied by western blot and immunohistochemistry, with results demonstrating that tumor cells overexpress ALCAM. These findings strongly suggest the possibility that ALCAM may have an important role in thyroid tumor biology. PMID:21364949

Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila.

PubMed

Morash, Michael G; Brassinga, Ann Karen C; Warthan, Michelle; Gourabathini, Poornima; Garduño, Rafael A; Goodman, Steven D; Hoffman, Paul S

2009-04-01

Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

PubMed Central

McKenzie, Jennifer A.; Buettmann, Evan G.; Gardner, Michael J.; Silva, Matthew J.

2015-01-01

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2. PMID:26344756

c-kit expression profile and regulatory factors during spermatogonial stem cell differentiation

PubMed Central

2013-01-01

Background It has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation. Results We have found that there are more than 4 transcripts of c-kit expressed in the cell lines and in the testes. The transcripts can be divided into short and long categories. The long transcripts include the full-length canonical c-kit transcript and the 3′ end short transcript. Short transcripts include the 3.4 kb short transcript and several truncated transcripts (1.9-3.2 kb). In addition, the 3.4 kb transcript (starting from intron 9 and covering exons 10 ~ 21) is discovered to be specifically expressed in the spermatogonia. The extracellular domain of Kit is obtained in the spermatogonia stage, but the intracellular domain (50 kDa) is constantly expressed in both SSCs and spermatogonia. The c-kit expression profiles in the testis and the spermatogonial stem cell lines vary after RA stimulation. The wave-like changes of the quantitative expression pattern of c-kit (increase initially and decrease afterwards) during the induction process are similar to that of the in vivo male germ cell development process. Conclusions There are dynamic transcription and translation changes of c-kit before and after SSCs’ anticipated differentiation and most importantly, RA is a significant upstream regulatory factor for c-kit expression. PMID:24161026

Efficient generation of functional CFTR-expressing airway epithelial cells from human pluripotent stem cells.

PubMed

Wong, Amy P; Chin, Stephanie; Xia, Sunny; Garner, Jodi; Bear, Christine E; Rossant, Janet

2015-03-01

Airway epithelial cells are of great interest for research on lung development, regeneration and disease modeling. This protocol describes how to generate cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR)-expressing airway epithelial cells from human pluripotent stem cells (PSCs). The stepwise approach from PSC culture to differentiation into progenitors and then mature epithelia with apical CFTR activity is outlined. Human PSCs that were inefficient at endoderm differentiation using our previous lung differentiation protocol were able to generate substantial lung progenitor cell populations. Augmented CFTR activity can be observed in all cultures as early as at 35 d of differentiation, and full maturation of the cells in air-liquid interface cultures occurs in <5 weeks. This protocol can be used for drug discovery, tissue regeneration or disease modeling.

Nanoarchitectured electrochemical cytosensors for selective detection of leukemia cells and quantitative evaluation of death receptor expression on cell surfaces.

PubMed

Zheng, Tingting; Fu, Jia-Ju; Hu, Lihui; Qiu, Fan; Hu, Minjin; Zhu, Jun-Jie; Hua, Zi-Chun; Wang, Hui

2013-06-04

The variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.

Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

PubMed

Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

2017-10-06

Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF.

PubMed

Jääskeläinen, Kirsi M; Plyusnina, Angelina; Lundkvist, Ake; Vaheri, Antti; Plyusnin, Alexander

2008-01-11

The competitiveness of two Tula hantavirus (TULV) isolates, TULV/Lodz and TULV/Moravia, was evaluated in interferon (IFN) -competent and IFN-deficient cells. The two isolates differ in the length of the open reading frame (ORF) encoding the nonstructural protein NSs, which has previously been shown to inhibit IFN response in infected cells. In IFN-deficient Vero E6 cells both TULV isolates survived equally well. In contrast, in IFN-competent MRC5 cells TULV/Lodz isolate, that possesses the NSs ORF for the full-length protein of 90 aa, survived for more consequent passages than TULV/Moravia isolate, which contains the ORF for truncated NSs protein (66-67 aa). Our data show that expression of a full-length NSs protein is beneficial for the virus survival and competitiveness in IFN-competent cells and not essential in IFN-deficient cells. These results suggest that the N-terminal aa residues are important for the full activity of the NSs protein.

A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

PubMed

Castillo, Andres; Wang, Lu; Koriyama, Chihaya; Eizuru, Yoshito; Jordan, King; Akiba, Suminori

2014-10-01

Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

Expression, purification, and characterization of recombinant human and murine milk fat globule-epidermal growth factor-factor 8.

PubMed

Castellanos, Erick R; Ciferri, Claudio; Phung, Wilson; Sandoval, Wendy; Matsumoto, Marissa L

2016-08-01

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVβ3 and αVβ5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVβ3 and αVβ5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8. Copyright © 2016 Elsevier Inc. All rights reserved.

[Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells].

PubMed

Wang, P; Liu, S; Cheng, B; Wu, X Z; Ding, S S; Xu, L; Liu, Y; Duan, L; Sun, S Z

2017-03-08

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

Brain-derived neurotrophic factor and its receptors in Bergmann glia cells.

PubMed

Poblete-Naredo, Irais; Guillem, Alain M; Juárez, Claudia; Zepeda, Rossana C; Ramírez, Leticia; Caba, Mario; Hernández-Kelly, Luisa C; Aguilera, José; López-Bayghen, Esther; Ortega, Arturo

2011-12-01

Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.

PubMed

Rees, D A; Hepburn, P J; McNicol, A M; Francis, K; Jasani, B; Lewis, M D; Farrell, W E; Lewis, B M; Scanlon, M F; Ham, J

2002-03-28

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.

Genomic profiling of rice sperm cell transcripts reveals conserved and distinct elements in the flowering plant male germ lineage.

PubMed

Russell, Scott D; Gou, Xiaoping; Wong, Chui E; Wang, Xinkun; Yuan, Tong; Wei, Xiaoping; Bhalla, Prem L; Singh, Mohan B

2012-08-01

Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field-grown, disease-free plants and RNA was processed for use with the full-genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10,732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin-mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome-wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self-regulating through highly up-regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post-fertilization expression of early embryo and (or) endosperm development. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Efficient, Glucose Responsive, and Islet-Specific Transgene Expression by a Modified Rat Insulin Promoter

PubMed Central

Chai, Renjie; Chen, Shuyuan; Ding, Jiahuan; Grayburn, Paul A

2009-01-01

This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various rat insulin promoter lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells, and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is 5-fold more active in INS-1 cells than the full length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent alpha cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified rat insulin promoter, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas. PMID:19727136

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

EpCAM and the biology of hepatic stem/progenitor cells

PubMed Central

Theise, Neil D.; Schmelzer, Eva; Boulter, Luke; Gires, Olivier; van Grunsven, Leo A.

2014-01-01

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration. PMID:25477371

High-throughput full-length single-cell mRNA-seq of rare cells.

PubMed

Ooi, Chin Chun; Mantalas, Gary L; Koh, Winston; Neff, Norma F; Fuchigami, Teruaki; Wong, Dawson J; Wilson, Robert J; Park, Seung-Min; Gambhir, Sanjiv S; Quake, Stephen R; Wang, Shan X

2017-01-01

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals

PubMed Central

Arenas-Mena, Cesar; Coffman, James A.

2016-01-01

Summary It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency. PMID:26173445

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

PubMed Central

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

2016-01-01

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

PubMed

Davidson, E J; Morris, L S; Scott, I S; Rushbrook, S M; Bird, K; Laskey, R A; Wilson, G E; Kitchener, H C; Coleman, N; Stern, P L

2003-01-27

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

PubMed

Dinculescu, Astra; Stupay, Rachel M; Deng, Wen-Tao; Dyka, Frank M; Min, Seok-Hong; Boye, Sanford L; Chiodo, Vince A; Abrahan, Carolina E; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W Clay; Hauswirth, William W

2016-01-01

Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

Most Influenza A Virions Fail To Express at Least One Essential Viral Protein

PubMed Central

Brooke, Christopher B.; Ince, William L.; Wrammert, Jens; Ahmed, Rafi; Wilson, Patrick C.; Bennink, Jack R.

2013-01-01

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population. PMID:23283949

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

PubMed

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Assessment and optimization of theileria parva sporozoite full-length p67 antigen expression in mammalian cells

USDA-ARS?s Scientific Manuscript database

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant vers...

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

PubMed

Testa, Jacqueline E; Chrastina, Adrian; Oh, Phil; Li, Yan; Witkiewicz, Halina; Czarny, Malgorzata; Buss, Tim; Schnitzer, Jan E

2009-08-01

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression.

PubMed

Pankow, R; Dürkop, H; Latza, U; Krause, H; Kunzendorf, U; Pohl, T; Bulfone-Paus, S

2000-07-01

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.

Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells

PubMed Central

2012-01-01

Background We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cells after immunization with T-cell-dependent antigen. Zizimin2 was revealed to be a new family member of Dock (dedicator of cytokinesis), Dock11, which is the guanine nucleotide exchange factor for Cdc42, a low-molecular-weight GTPase. However, the molecular function of Zizimin2 in acquired immunity has not been elucidated. Results In this study, we show that the protein expression of Zizimin2, which is also restricted to lymphoid tissues and lymphocytes, is reduced in aged mice. Over-expression of full-length Zizimin2 induced filopodial formation in 293T cells, whereas expression of CZH2 domain inhibited it. Stimulation of Fcγ receptor and Toll-like receptor 4 triggered Zizimin2 up-regulation and Cdc42 activation in bone marrow-derived dendritic cells. Conclusions These data suggest that Zizimin2 is an immune-related and age-regulated guanine nucleotide exchange factor, which facilitates filopodial formation through activation of Cdc42, which results in activation of cell migration. PMID:22494997

RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

DOE Office of Scientific and Technical Information (OSTI.GOV)

López, Claudia S., E-mail: lopezcl@ohsu.edu; Sloan, Rachel; Cylinder, Isabel

The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag proteinmore » expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.« less

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

PubMed

Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

2014-03-01

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon.

PubMed

Dyment, Nathaniel A; Kazemi, Namdar; Aschbacher-Smith, Lindsey E; Barthelery, Nicolas J; Kenter, Keith; Gooch, Cynthia; Shearn, Jason T; Wylie, Christopher; Butler, David L

2012-01-01

Tendon injuries are major orthopedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development. Copyright © 2011 Orthopaedic Research Society.

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

PubMed

Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

2015-01-01

The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

2015-04-01

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope.more » Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.« less

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression.

PubMed

Irioda, Ana Carolina; Cassilha, Rafael; Zocche, Larissa; Francisco, Julio Cesar; Cunha, Ricardo Correa; Ferreira, Priscila Elias; Guarita-Souza, Luiz Cesar; Ferreira, Reginaldo Justino; Mogharbel, Bassam Felipe; Garikipati, Venkata Naga Srikanth; Souza, Daiany; Beltrame, Mirian Perlingeiro; de Carvalho, Katherine Athayde Teixeira

2016-01-01

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

Association between Twist and multidrug resistance gene-associated proteins in Taxol®-resistant MCF-7 cells and a 293 cell model of Twist overexpression.

PubMed

Wang, Li; Tan, Rui-Zhi; Zhang, Zhi-Xia; Yin, Rui; Zhang, Yong-Liang; Cui, Wei-Jia; He, Tao

2018-01-01

Multidrug resistance (MDR) severely limits the effectiveness of chemotherapy. Previous studies have identified Twist as a key factor of acquired MDR in breast, gastric and prostate cancer. However, the underlying mechanisms of action of Twist in MDR remain unclear. In the present study, the expression levels of MDR-associated proteins, including lung resistance-related protein (LRP), topoisomerase IIα (TOPO IIα), MDR-associated protein (MRP) and P-glycoprotein (P-gp), and the expression of Twist in cancerous tissues and pericancerous tissues of human breast cancer, were examined. In order to simulate Taxol ® resistance in cells, a Taxol ® -resistant human mammary adenocarcinoma cell subline (MCF-7/Taxol ® ) was established by repeatedly exposing MCF-7 cells to high concentrations of Taxol ® (up to 15 µg/ml). Twist was also overexpressed in 293 cells by transfecting this cell line with pcDNA5/FRT/TO vector containing full-length hTwist cDNA to explore the dynamic association between Twist and MDR gene-associated proteins. It was identified that the expression levels of Twist, TOPO IIα, MRP and P-gp were upregulated and LRP was downregulated in human breast cancer tissues, which was consistent with the expression of these proteins in the Taxol ® -resistant MCF-7 cell model. Notably, the overexpression of Twist in 293 cells increased the resistance to Taxol ® , Trichostatin A and 5-fluorouracil, and also upregulated the expression of MRP and P-gp. Taken together, these data demonstrated that Twist may promote drug resistance in cells and cancer tissues through regulating the expression of MDR gene-associated proteins, which may assist in understanding the mechanisms of action of Twist in drug resistance.

Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans

PubMed Central

Sierra, Crystal S.; Haase, Steven B.

2016-01-01

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation. PMID:27918582

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jambaldorj, Jamiyansuren; Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine, Yamagata 990-9585; Central Scientific Research Laboratory, Institute of Medical Sciences, Ulaanbaatar

2012-08-24

Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1),more » which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.« less

Cooperation of hTERT, SV40 T Antigen and Oncogenic Ras in Tumorigenesis: A Cell Transplantation Model Using Bovine Adrenocortical Cells1

PubMed Central

Thomas, Michael; Suwa, Tetsuya; Yang, Lianqing; Zhao, Lifang; Hawks, Christina L; Hornsby, Peter J

2002-01-01

Abstract Expression of TERT, the reverse transcriptase component of telomerase, is necessary to convert normal human cells to cancer cells. Despite this, “telomerization” by hTERT does not appear to alter the normal properties of cells. In a cell transplantation model in which bovine adrenocortical cells form vascularized tissue structures beneath the kidney capsule in scid mice, telomerization does not perturb the functional tissue-forming capacity of the cells. This cell transplantation model was used to study the cooperation of hTERT with SV40 T antigen (SV40 TAg) and oncogenic Ras in tumorigenesis. Only cells expressing all three genes were tumorigenic; this required large T, but not small t, antigen. These cells produced a continuously expanding tissue mass; they were invasive with respect to adjacent organs and eventually destroyed the kidney. Cells expressing only hTERT or only Ras produced minimally altered tissues. In contrast, SV40 TAg alone produced noninvasive nodules beneath the kidney capsule that had high proliferation rates balanced by high rates of apoptosis. The use of cell transplantation techniques in a cell type that is able to form tissue structures with or without full neoplastic conversion allows the phenotypes produced by individual cooperating oncogenes to be observed. PMID:12407443

A Splice Variant of the Human Ion Channel TRPM2 Modulates Neuroblastoma Tumor Growth through Hypoxia-inducible Factor (HIF)-1/2α*

PubMed Central

Chen, Shu-jen; Hoffman, Nicholas E.; Shanmughapriya, Santhanam; Bao, Lei; Keefer, Kerry; Conrad, Kathleen; Merali, Salim; Takahashi, Yoshinori; Abraham, Thomas; Hirschler-Laszkiewicz, Iwona; Wang, JuFang; Zhang, Xue-Qian; Song, Jianliang; Barrero, Carlos; Shi, Yuguang; Kawasawa, Yuka Imamura; Bayerl, Michael; Sun, Tianyu; Barbour, Mustafa; Wang, Hong-Gang; Madesh, Muniswamy; Cheung, Joseph Y.; Miller, Barbara A.

2014-01-01

The calcium-permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full-length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of forkhead box transcription factor 3a (FOXO3a) and superoxide dismutase 2. Cells expressing the dominant negative short isoform (TRPM2-S) had reduced FOXO3a and superoxide dismutase 2 levels, reduced calcium influx in response to oxidative stress, and enhanced reactive oxygen species, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared with tumors expressing TRPM2-L. Expression of hypoxia-inducible factor (HIF)-1/2α was significantly reduced in TRPM2-S-expressing tumor cells as was expression of target proteins regulated by HIF-1/2α including those involved in glycolysis (lactate dehydrogenase A and enolase 2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (cytochrome oxidase 4.1/4.2 in complex IV). The reduction in HIF-1/2α was mediated through both significantly reduced HIF-1/2α mRNA levels and increased levels of von Hippel-Lindau E3 ligase in TRPM2-S-expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S-expressing cells after doxorubicin treatment was rescued by gain of HIF-1 or -2α function. These data suggest that TRPM2 activity is important for tumor growth and for cell viability and survival following doxorubicin treatment and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2α, mitochondrial function, and mitophagy. PMID:25391657

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

PubMed

Espana, Edgar M; Di Pascuale, Mario A; He, Hua; Kawakita, Tetsuya; Raju, Vadrevu K; Liu, Chia-Yang; Tseng, Scheffer C G

2004-09-01

To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker. Copyright Association for Research in Vision and Ophthalmology

Drug transporter gene expression in human colorectal tissue and cell lines: modulation with antiretrovirals for microbicide optimization.

PubMed

Mukhopadhya, Indrani; Murray, Graeme I; Berry, Susan; Thomson, John; Frank, Bruce; Gwozdz, Garry; Ekeruche-Makinde, Julia; Shattock, Robin; Kelly, Charles; Iannelli, Francesco; Pozzi, Gianni; El-Omar, Emad M; Hold, Georgina L; Hijazi, Karolin

2016-02-01

The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

PubMed

Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

2013-08-15

Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Pharmacological selectivity of the cloned human P2U-purinoceptor: potent activation by diadenosine tetraphosphate.

PubMed Central

Lazarowski, E. R.; Watt, W. C.; Stutts, M. J.; Boucher, R. C.; Harden, T. K.

1995-01-01

1. The human P2U-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2. High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3. UTP, ATP and ATP gamma S were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 microM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, alpha,beta-MeATP, and 2MeSADP, respectively. 4. Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2U-purinoceptors. PMID:8564228

Differential induction of CD94 and NKG2 in CD4 helper T cells. A consequence of influenza virus infection and interferon-γ?

PubMed Central

Graham, Christine M; Christensen, Jillian R; Thomas, D Brian

2007-01-01

Influenza A virus causes worldwide epidemics and pandemics and the investigation of memory T helper (Th) cells that help maintain serological memory following infection is important for vaccine design. In this study we investigated CD94 and NKG2 gene expression in memory CD4 T-cell clones established from the spleens of C57BL/10 (H-2b) and BALB/c (H-2d) mice infected with influenza A virus (H3N2). CD94 and NKG2A/C/E proteins form heterodimeric membrane receptors that are involved in virus recognition. CD94 and NKG2 expression have been well characterized in natural killer (NK) and cytotoxic T cells. Despite CD94 being potentially an important marker for Th1 cells involved in virus infection, however, there has been little investigation of its expression or function in the CD4 T-cell lineage and no studies have looked at in-vivo-generated Th cells or memory cells. We show in this study that in-vivo-generated CD4 Th1 cells, but not Th2 cells, exhibited full-length CD94 and NKG2A gene expression following activation with viral peptide. For NKG2A, a novel ‘short’ (possibly redundant) truncated isoform was detectable in a Th2 cell clone. Another member of the NK receptor family, NKG2D, but not NKG2C or E, was also differentially expressed in Th1 cells. We show here that CD94 and NKG2A may exist as multiple isoforms with the potential to distinguish helper T-cell subsets. PMID:17462078

Tau Fibril Formation in Cultured Cells Compatible with a Mouse Model of Tauopathy.

PubMed

Matsumoto, Gen; Matsumoto, Kazuki; Kimura, Taeko; Suhara, Tetsuya; Higuchi, Makoto; Sahara, Naruhiko; Mori, Nozomu

2018-05-17

Neurofibrillary tangles composed of hyperphosphorylated tau protein are primarily neuropathological features of a number of neurodegenerative diseases collectively termed tauopathy. To understand the mechanisms underlying the cause of tauopathy, precise cellular and animal models are required. Recent data suggest that the transient introduction of exogenous tau can accelerate the development of tauopathy in the brains of non-transgenic and transgenic mice expressing wild-type human tau. However, the transmission mechanism leading to tauopathy is not fully understood. In this study, we developed cultured-cell models of tauopathy representing a human tauopathy. Neuro2a (N2a) cells containing propagative tau filaments were generated by introducing purified tau fibrils. These cell lines expressed full-length (2N4R) human tau and the green fluorescent protein (GFP)-fused repeat domain of tau with P301L mutation. Immunocytochemistry and super-resolution microscopic imaging revealed that tau inclusions exhibited filamentous morphology and were composed of both full-length and repeat domain fragment tau. Live-cell imaging analysis revealed that filamentous tau inclusions are transmitted to daughter cells, resulting in yeast-prion-like propagation. By a standard method of tau preparation, both full-length tau and repeat domain fragments were recovered in sarkosyl insoluble fraction. Hyperphosphorylation of full-length tau was confirmed by the immunoreactivity of phospho-Tau antibodies and mobility shifts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These properties were similar to the biochemical features of P301L mutated human tau in a mouse model of tauopathy. In addition, filamentous tau aggregates in cells barely co-localized with ubiquitins, suggesting that most tau aggregates were excluded from protein degradation systems, and thus propagated to daughter cells. The present cellular model of tauopathy will provide an advantage for dissecting the mechanisms of tau aggregation and degradation and be a powerful tool for drug screening to prevent tauopathy.

Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Jon M.; Diamond, Deborah L.; Chan, Eric Y.

2005-06-01

The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled tomore » mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 {micro}g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.« less

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche

PubMed Central

2018-01-01

ABSTRACT Stem cells reside in a niche, a local environment whose cellular and molecular complexity is still being elucidated. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap cell-produced Wnt6 survival signal. PMID:29361569

Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche.

PubMed

Wang, Xiaoxi; Page-McCaw, Andrea

2018-02-07

Stem cells reside in a niche, a local environment whose cellular and molecular complexity is still being elucidated. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap cell-produced Wnt6 survival signal. © 2018. Published by The Company of Biologists Ltd.

Dysregulation of Ezrin phosphorylation prevents metastasis and alters cellular metabolism in osteosarcoma

PubMed Central

Ren, Ling; Hong, Sung-Hyeok; Chen, Qing-Rong; Briggs, Joseph; Cassavaugh, Jessica; Srinivasan, Satish; Lizardo, Michael M.; Mendoza, Arnulfo; Xia, Ashley Y.; Avadhani, Narayan; Khan, Javed; Khanna, Chand

2013-01-01

Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers (1, 2), however, the precise mechanistic basis for its role remains unknown. Here we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biological characteristics to full length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism. PMID:22147261

Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression.

PubMed

Kameda, H; Risinger, J I; Han, B B; Baek, S J; Barrett, J C; Abe, T; Takeuchi, T; Glasgow, W C; Eling, T E

2001-10-01

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

PubMed Central

Marini, Pietro; Cascio, Maria-Grazia; King, Angela; Pertwee, Roger G; Ross, Ruth A

2013-01-01

Background and Purpose Although cannabinoid CB2 receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2-transfected CHO cells. Experimental Approach We tested the ability of well-known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2-transfected CHO cell membranes. cAMP assays were also performed in hCB2-CHO cells. Key Results The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2-CHO cells, in both [35S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB2-CHO cells when tested in the [35S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB2-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB2-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues. Conclusion and Implications Our results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution. PMID:23711022

Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments.

PubMed Central

Nutt, S L; Morrison, A M; Dörfler, P; Rolink, A; Busslinger, M

1998-01-01

The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre-BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5-deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig-alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre-BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF-1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context. PMID:9545244

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice.

PubMed

Taghiyar, Leila; Hesaraki, Mahdi; Sayahpour, Forough Azam; Satarian, Leila; Hosseini, Samaneh; Aghdami, Naser; Baghaban Eslaminejad, Mohamadreza

2017-06-23

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox ( Msx ) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx -regulated genes ( Bmp4 , Fgf8 , and keratin 14 ( K14 )) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx -overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8 , and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx -transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor-specific chimeric antigen receptor.

PubMed

Krug, Christian; Wiesinger, Manuel; Abken, Hinrich; Schuler-Thurner, Beatrice; Schuler, Gerold; Dörrie, Jan; Schaft, Niels

2014-10-01

Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 10(8) CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.

Transient receptor potential vanilloid type 2 (TRPV2) expression in normal urothelium and in urothelial carcinoma of human bladder: correlation with the pathologic stage.

PubMed

Caprodossi, Sara; Lucciarini, Roberta; Amantini, Consuelo; Nabissi, Massimo; Canesin, Giacomo; Ballarini, Patrizia; Di Spilimbergo, Adriana; Cardarelli, Marco Andrea; Servi, Lucilla; Mammana, Gabriele; Santoni, Giorgio

2008-09-01

To evaluate the expression of transient receptor potential vanilloid type 2 (TRPV2) in normal human bladder and urothelial carcinoma (UC) tissues. Bladder specimens were obtained by transurethral resection or radical cystectomy. TRPV2 mRNA expression in normal human urothelial cells (NHUCs), UC cell lines, and formalin-fixed paraffin-embedded normal (n=6) and cancer bladder tissues (n=58) was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-PCR). TRPV2 protein expression was assessed by cytofluorimetric and confocal microscopy analyses in NHUCs and UC cells and by Western blotting and immunohistochemistry in normal and UC tissues. Enhanced TRPV2 mRNA and protein expression was found in high-grade and -stage UC specimens and UC cell lines. Both the full-length TRPV2 (hTRPV2) and a short splice-variant (s-TRPV2) were detected in NHUC and normal bladder specimens, whereas a progressive decline of s-TRPV2 in pTa, pT1, and pT2 stages was observed, up to a complete loss in pT3 and pT4 UC specimens. Normal human urothelial cells and bladder tissue specimens express TRPV2 at both the mRNA and protein levels. A progressive loss of s-TRPV2 accompanied by a marked increase of hTRPV2 expression was found in high-grade and -stage UC tissues.

piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

PubMed

Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

2016-01-29

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

PubMed

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

Fibroblast growth factor receptor inhibition induces loss of matrix MCL1 and necrosis in cholangiocarcinoma.

PubMed

Kabashima, Ayano; Hirsova, Petra; Bronk, Steven F; Hernandez, Matthew C; Truty, Mark J; Rizvi, Sumera; Kaufmann, Scott H; Gores, Gregory J

2018-03-08

Myeloid cell leukemia 1 (MCL1), a prosurvival member of the BCL2 protein family, has a pivotal role in human cholangiocarcinoma (CCA) cell survival. We previously reported that fibroblast growth factor receptor (FGFR) signalling mediates MCL1-dependent survival of CCA cells in vitro and in vivo. However, the mode and mechanisms of cell death in this model were not delineated. Human CCA cell lines were treated with the pan-FGFR inhibitor LY2874455 and the mode of cell death examined by several complementary assays. Mitochondrial oxidative metabolism was examined using a XF24 extracellular flux analyser. The efficiency of FGFR inhibition in patient-derived xenografts (PDX) was also assessed. CCA cells expressed two species of MCL1, a full-length form localised to the outer mitochondrial membrane, and an N terminus-truncated species compartmentalised within the mitochondrial matrix. The pan-FGFR inhibitor LY2874455 induced non-apoptotic cell death in the CCA cell lines associated with cellular depletion of both MCL1 species. The cell death was accompanied by failure of mitochondrial oxidative metabolism and was most consistent with necrosis. Enforced expression of N terminus-truncated MCL1 targeted to the mitochondrial matrix, but not full-length MCL1 targeted to the outer mitochondrial membrane, rescued cell death and mitochondrial function. LY2874455 treatment of PDX-bearing mice was associated with tumour cell loss of MCL1 and cell necrosis. FGFR inhibition induces loss of matrix MCL1, resulting in cell necrosis. These observations support a heretofore unidentified, alternative MCL1 survival function, namely prevention of cell necrosis, and have implications for treatment of human CCA. Herein, we report that therapeutic inhibition of a cell receptor expressed by bile duct cancer cells resulted in the loss of a critical survival protein termed MCL1. Cellular depletion of MCL1 resulted in the death of the cancer cells by a process characterised by cell rupture. Cell death by this process can stimulate the immune system and has implications for combination therapy using receptor inhibition with immunotherapy. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Targeting Breast Cancer Vasculature

DTIC Science & Technology

2006-03-01

E., and Hanahan, D. Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 4:393...expressing full-length myc-tagged metadherin, myc-vimen- tin, or myc-pCMV were analyzed by flow cytometry . Anti-myc antibodies were applied to the cells...In 4T1 tumor cell extracts, anti-metadherin(378-440) detectedthen stained with anti-myc antibodies. Using flow cytometry , proteins with apparent

Growth and gene expression are predominantly controlled by distinct regions of the human IL-4 receptor.

PubMed

Ryan, J J; McReynolds, L J; Keegan, A; Wang, L H; Garfein, E; Rothman, P; Nelms, K; Paul, W E

1996-02-01

IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

Matrix-Dependent Regulation of AKT in Hepsin-Overexpressing PC3 Prostate Cancer Cells12

PubMed Central

Wittig-Blaich, Stephanie M; Kacprzyk, Lukasz A; Eismann, Thorsten; Bewerunge-Hudler, Melanie; Kruse, Petra; Winkler, Eva; Strauss, Wolfgang S L; Hibst, Raimund; Steiner, Rudolf; Schrader, Mark; Mertens, Daniel; Sültmann, Holger; Wittig, Rainer

2011-01-01

The serine-protease hepsin is one of the most prominently overexpressed genes in human prostate carcinoma. Forced expression of the enzyme in mice prostates is associated with matrix degradation, invasive growth, and prostate cancer progression. Conversely, hepsin overexpression in metastatic prostate cancer cell lines was reported to induce cell cycle arrest and reduction of invasive growth in vitro. We used a system for doxycycline (dox)-inducible target gene expression in metastasis-derived PC3 cells to analyze the effects of hepsin in a quantitative manner. Loss of viability and adhesion correlated with hepsin expression levels during anchorage-dependent but not anchorage-independent growth. Full expression of hepsin led to cell death and detachment and was specifically associated with reduced phosphorylation of AKT at Ser473, which was restored by growth on matrix derived from RWPE1 normal prostatic epithelial cells. In the chorioallantoic membrane xenograft model, hepsin overexpression in PC3 cells reduced the viability of tumors but did not suppress invasive growth. The data presented here provide evidence that elevated levels of hepsin interfere with cell adhesion and viability in the background of prostate cancer as well as other tissue types, the details of which depend on the microenvironment provided. Our findings suggest that overexpression of the enzyme in prostate carcinogenesis must be spatially and temporally restricted for the efficient development of tumors and metastases. PMID:21750652

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

PubMed

Blomster, Linda V; Strøbaek, Dorte; Hougaard, Charlotte; Klein, Jessica; Pinborg, Lars H; Mikkelsen, Jens D; Christophersen, Palle

2016-12-01

The K Ca 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of K Ca 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a K Ca 3.1/K Ca 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective K Ca 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed K Ca 3.1, and the difference in current between full activation and inhibition (ΔK Ca 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial K Ca 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the K Ca 3.1 current nor the fraction of K Ca 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the K Ca 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the K Ca 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a K ir channel. The high functional expression of K Ca 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078. © 2016 Wiley Periodicals, Inc.

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

PubMed

Mead, Emma J; Chiverton, Lesley M; Spurgeon, Sarah K; Martin, Elaine B; Montague, Gary A; Smales, C Mark; von der Haar, Tobias

2012-01-01

Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

Heat Shock Protein-90 Inhibitors Enhance Antigen Expression on Melanomas and Increase T Cell Recognition of Tumor Cells

PubMed Central

Haggerty, Timothy J.; Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Pandolfi, Franco; Kurnick, James T.

2014-01-01

In an effort to enhance antigen-specific T cell recognition of cancer cells, we have examined numerous modulators of antigen-expression. In this report we demonstrate that twelve different Hsp90 inhibitors (iHsp90) share the ability to increase the expression of differentiation antigens and MHC Class I antigens. These iHsp90 are active in several molecular and cellular assays on a series of tumor cell lines, including eleven human melanomas, a murine B16 melanoma, and two human glioma-derived cell lines. Intra-cytoplasmic antibody staining showed that all of the tested iHsp90 increased expression of the melanocyte differentiation antigens Melan-A/MART-1, gp100, and TRP-2, as well as MHC Class I. The gliomas showed enhanced gp100 and MHC staining. Quantitative analysis of mRNA levels showed a parallel increase in message transcription, and a reporter assay shows induction of promoter activity for Melan-A/MART-1 gene. In addition, iHsp90 increased recognition of tumor cells by T cells specific for Melan-A/MART-1. In contrast to direct Hsp90 client proteins, the increased levels of full-length differentiation antigens that result from iHsp90 treatment are most likely the result of transcriptional activation of their encoding genes. In combination, these results suggest that iHsp90 improve recognition of tumor cells by T cells specific for a melanoma-associated antigen as a result of increasing the expressed intracellular antigen pool available for processing and presentation by MHC Class I, along with increased levels of MHC Class I itself. As these Hsp90 inhibitors do not interfere with T cell function, they could have potential for use in immunotherapy of cancer. PMID:25503774

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

PGE2 through the EP4 receptor controls smooth muscle gene expression patterns in the ductus arteriosus critical for remodeling at birth

PubMed Central

Gruzdev, Artiom; Nguyen, MyTrang; Kovarova, Martina; Koller, Beverly H.

2012-01-01

The ductus arteriosus (DA) is a fetal shunt that directs right ventricular outflow away from pulmonary circulation and into the aorta. Critical roles for prostaglandin E2 (PGE2) and the EP4 receptor (EP4) have been established in maintaining both the patency of the vessel in utero and in its closure at birth. Here we have generated mice in which loss of EP4 expression is limited to either the smooth muscle (SMC) or endothelial cells and demonstrated that SMC, but not endothelial cell expression of EP4 is required for DA closure. The genome wide expression analysis of full term wild type and EP4−/− DA indicates that PGE2/EP4 signaling modulates expression of a number of unique pathways, including those involved in SMC proliferation, cell migration, and vascular tone. Together this supports a mechanism by which maturation and increased contractility of the vessel is coupled to the potent smooth muscle dilatory actions of PGE2. PMID:22342504

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

PubMed Central

Wong, Darice Y.; Ranganath, Thanmayi; Kasko, Andrea M.

2015-01-01

Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying. PMID:26418040

Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines.

PubMed

Zhao, Wei-Xiu; Zhuang, Xu; Huang, Tao-Tao; Feng, Ran; Lin, Jian-Hua

2015-01-01

To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR. Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit. We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated. Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.

Characterisation and expression of myogenesis regulatory factors during in vitro myoblast development and in vivo fasting in the gilthead sea bream (Sparus aurata).

PubMed

García de la serrana, Daniel; Codina, Marta; Capilla, Encarnación; Jiménez-Amilburu, Vanesa; Navarro, Isabel; Du, Shao-Jun; Johnston, Ian A; Gutiérrez, Joaquim

2014-01-01

The aim of this study was to characterise a primary cell culture isolated from fast skeletal muscle of the gilthead sea bream. Gene expression profiles during culture maturation were compared with those obtained from a fasting-refeeding model which is widely used to modulate myogenesis in vivo. Myogenesis is controlled by numerous extracellular signals together with intracellular transcriptional factors whose coordinated expression is critical for the appropriate development of muscle fibres. Full-length cDNAs for the transcription factors Myf5, Mrf4, Pax7 and Sox8 were cloned and sequenced for gilthead sea bream. Pax7, sox8, myod2 and myf5 levels were up-regulated during the proliferating phase of the myogenic cultures coincident with the highest expression of proliferating cell nuclear antigen (PCNA). In contrast, myogenin and mrf4 transcript abundance was highest during the differentiation phase of the culture when myotubes were present, and was correlated with increased myosin heavy chain (mhc) and desmin expression. In vivo, 30days of fasting resulted in muscle fibre atrophy, a reduction in myod2, myf5 and igf1 expression, lower number of Myod-positive cells, and decreased PCNA protein expression, whereas myogenin expression was not significantly affected. Myostatin1 (mstn1) and pax7 expression were up-regulated in fasted relative to well-fed individuals, consistent with a role for Pax7 in the reduction of myogenic cell activity with fasting. The primary cell cultures and fasting-feeding experiments described provide a foundation for the future investigations on the regulation of muscle growth in gilthead sea bream. © 2013.

Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets

PubMed Central

Pensieroso, Simone; Tolazzi, Monica; Chiappetta, Stefania; Nozza, Silvia; Lazzarin, Adriano; Tambussi, Giuseppe; Scarlatti, Gabriella

2015-01-01

Introduction During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART). Materials and Methods To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation. Results Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups. Conclusions In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI. PMID:26474181

Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets.

PubMed

Pogliaghi, Manuela; Ripa, Marco; Pensieroso, Simone; Tolazzi, Monica; Chiappetta, Stefania; Nozza, Silvia; Lazzarin, Adriano; Tambussi, Giuseppe; Scarlatti, Gabriella

2015-01-01

During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART). To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation. Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups. In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI.

N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase.

PubMed

Navarre, Catherine; Smargiasso, Nicolas; Duvivier, Laurent; Nader, Joseph; Far, Johann; De Pauw, Edwin; Boutry, Marc

2017-06-01

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

Silencing alpha-fetoprotein inhibits VEGF and MMP-2/9 production in human hepatocellular carcinoma cell.

PubMed

Meng, Wenbo; Li, Xun; Bai, Zhongtian; Li, Yan; Yuan, Jinqiu; Liu, Tao; Yan, Jun; Zhou, Wence; Zhu, Kexiang; Zhang, Hui; Li, Yumin

2014-01-01

Alpha-fetoprotein not only serves as a diagnostic marker for liver cancer, but also posses a variety of biological functions. However, the role of Alpha-fetoprotein on tumor angiogenesis and cell invasion remains incompletely understood. In this study, we aimed to evaluate if Alpha-fetoprotein can regulate the major angiogenic factors and matrix metalloproteinases in human liver cancer cells. Alpha-fetoprotein silencing was achieved by Stealth RNAi. Expression of Alpha-fetoprotein was examined by a full-automatic electrochemistry luminescence immunity analyzer. Expression of VEGF, VEGFR-2, MMP-9, and MMP-2 was examined by Western blot and immunocytochemistry. Apoptosis was detected by TUNEL assay. Angiogenesis was detected by in vitro angiogenesis assay kit. Silencing of Alpha-fetoprotein led to an increased apoptosis, which was associated with a decreased expression of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, matrix metalloproteinases-2/9. These results suggest that Alpha-fetoprotein may play a regulatory role on angiogenesis and cell invasion during liver cancer development.

T cell activation responses are differentially regulated during clinorotation and in spaceflight

NASA Technical Reports Server (NTRS)

Hashemi, B. B.; Penkala, J. E.; Vens, C.; Huls, H.; Cubbage, M.; Sams, C. F.

1999-01-01

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.

Prolonged Sox4 Expression in Oligodendrocytes Interferes with Normal Myelination in the Central Nervous System▿ †

PubMed Central

Potzner, Michaela R.; Griffel, Carola; Lütjen-Drecoll, Elke; Bösl, Michael R.; Wegner, Michael; Sock, Elisabeth

2007-01-01

The highly related transcription factors Sox4 and Sox11 are both expressed in oligodendrocyte precursors. Yet whether they have a function in oligodendrocyte development is unknown. By overexpressing Sox4 under the control of 3.1 kb of 5′ flanking sequences of the myelin basic protein gene in transgenic mice, we extended Sox4 expression in the oligodendrocyte lineage from oligodendrocyte precursors to cells undergoing terminal differentiation. As a consequence of transgene expression, mice develop the full spectrum of phenotypic traits associated with a severe hypomyelination during the first postnatal weeks. Myelin gene expression was severely reduced, and myelin dramatically thinned in several central nervous system (CNS) regions. Despite these disturbances in CNS myelination, the number of oligodendrocytic cells remained unaltered. Considering that apoptosis rates were normal and proliferation only slightly increased, oligodendrocytes likely persist in a premyelinating to early myelinating state. This shows that prolonged Sox4 expression in cells of the oligodendrocyte lineage is incompatible with the acquisition of a fully mature phenotype and argues that the presence of Sox4, and possibly Sox11, in oligodendrocyte precursors may normally prevent premature differentiation. PMID:17515609

Prolonged Sox4 expression in oligodendrocytes interferes with normal myelination in the central nervous system.

PubMed

Potzner, Michaela R; Griffel, Carola; Lütjen-Drecoll, Elke; Bösl, Michael R; Wegner, Michael; Sock, Elisabeth

2007-08-01

The highly related transcription factors Sox4 and Sox11 are both expressed in oligodendrocyte precursors. Yet whether they have a function in oligodendrocyte development is unknown. By overexpressing Sox4 under the control of 3.1 kb of 5' flanking sequences of the myelin basic protein gene in transgenic mice, we extended Sox4 expression in the oligodendrocyte lineage from oligodendrocyte precursors to cells undergoing terminal differentiation. As a consequence of transgene expression, mice develop the full spectrum of phenotypic traits associated with a severe hypomyelination during the first postnatal weeks. Myelin gene expression was severely reduced, and myelin dramatically thinned in several central nervous system (CNS) regions. Despite these disturbances in CNS myelination, the number of oligodendrocytic cells remained unaltered. Considering that apoptosis rates were normal and proliferation only slightly increased, oligodendrocytes likely persist in a premyelinating to early myelinating state. This shows that prolonged Sox4 expression in cells of the oligodendrocyte lineage is incompatible with the acquisition of a fully mature phenotype and argues that the presence of Sox4, and possibly Sox11, in oligodendrocyte precursors may normally prevent premature differentiation.

Heterogeneity of cellular proliferation within transitional cell carcinoma: correlation of protein kinase C alpha/betaI expression and activity.

PubMed

Aaltonen, Vesa; Koivunen, Jussi; Laato, Matti; Peltonen, Juha

2006-07-01

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Decreased Superoxide Production, Degranulation, Tumor Necrosis Factor Alpha Secretion, and CD11b/CD18 Receptor Expression by Adherent Monocytes from Preterm Infants

PubMed Central

Kaufman, David; Kilpatrick, Laurie; Hudson, R. Guy; Campbell, Donald E.; Kaufman, Ann; Douglas, Steven D.; Harris, Mary C.

1999-01-01

Preterm infants have an increased incidence of infection, which is principally due to deficiencies in neonatal host defense mechanisms. Monocyte adherence is important in localizing cells at sites of infection and is associated with enhanced antimicrobial functions. We isolated cord blood monocytes from preterm and full-term infants to study their adhesion and immune functions, including superoxide (O2−) generation, degranulation, and cytokine secretion and their adhesion receptors. O2− production and degranulation were significantly diminished, by 28 and 37%, respectively, in adherent monocytes from preterm infants compared to full-term infants (P < 0.05); however, these differences were not seen in freshly isolated cells. We also observed a significant decrease of 35% in tumor necrosis factor alpha secretion by lipopolysaccharide-stimulated adherent monocytes from preterm infants compared to full-term infants (P < 0.05); however, this difference was not observed in interleukin-1β or interleukin-6 production by the monocytes. The cell surface expression of the CD11b/CD18 adhesion receptor subunits was significantly decreased (by 60 and 52%, respectively) in monocytes from preterm infants compared to full-term infants (P < 0.01). The cascade of the immune response to infection involves monocyte upregulation and adherence via CD11b/CD18 receptors followed by cell activation and the release of cytokines and bactericidal products. We speculate that monocyte adherence factors may be important in the modulation of immune responses in preterm infants. PMID:10391855

Effects of FR235222, a novel HDAC inhibitor, in proliferation and apoptosis of human leukaemia cell lines: role of annexin A1.

PubMed

Petrella, Antonello; D'Acunto, Cosimo Walter; Rodriquez, Manuela; Festa, Michela; Tosco, Alessandra; Bruno, Ines; Terracciano, Stefania; Taddei, Maurizio; Paloma, Luigi Gomez; Parente, Luca

2008-03-01

FR235222, a novel histone deacetylase inhibitor (HDACi), at 50nM caused accumulation of acetylated histone H4, inhibition of cell proliferation and G1 cycle arrest accompanied by increase of p21 and down-regulation of cyclin E in human promyelocytic leukaemia U937 cells. The compound was also able to increase the protein and mRNA levels of annexin A1 (ANXA1) without effects on apoptosis. Similar effects were observed in human chronic myelogenous leukaemia K562 cells and human T cell leukaemia Jurkat cells. Cycle arrest and ANXA1 expression, without significant effects on apoptosis, were also induced by different HDACi like suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). FR235222 at 0.5 microM stimulated apoptosis of all leukaemia cell lines associated to an increased expression of the full-length (37kDa) protein and the appearance of a 33kDa N-terminal cleavage product in both cytosol and membrane. These results suggest that ANXA1 expression may mediate cycle arrest induced by low doses FR235222, whereas apoptosis induced by high doses FR235222 is associated to ANXA1 processing.

Lamin B receptor (LBR) regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: Implications for cholesterol biosynthesis in regulating myelopoiesis

PubMed Central

Subramanian, Gayathri; Chaudhury, Pulkit; Malu, Krishnakumar; Fowler, Samantha; Manmode, Rahul; Gotur, Deepali; Zwerger, Monika; Ryan, David; Roberti, Rita; Gaines, Peter

2011-01-01

Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin binding domains plus a C-terminal sterol Δ14 reductase domain. LBR expression increases during neutrophil differentiation and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (EPRO cells) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. Here we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EPRO cells, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ14 reductase domain of LBR plays a critical role in cholesterol biosynthesis, and that this process is essential to both myeloid cell growth and functional maturation. PMID:22140257

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

PubMed

Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0. 05). A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

In vivo regulation of Bcl6 and T follicular helper cell development1

PubMed Central

Poholek, Amanda C.; Hansen, Kyle; Hernandez, Sairy G.; Eto, Danelle; Chandele, Anmol; Weinstein, Jason S.; Dong, Xuemei; Odegard, Jared M.; Kaech, Susan M.; Dent, Alexander L.; Crotty, Shane; Craft, Joe

2010-01-01

Follicular helper T (TFH) cells, defined by expression of the surface markers CXCR5 and PD-1 and synthesis of IL-21, require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells, and the cytokines IL-6 and IL-21, in the in vivo regulation of Bcl6 expression and TFH cell development. We found that TFH cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand-1 (PSGL1, a CCL19- and CCL21-binding protein), indicating that, like CXCR5 and PD-1 upregulation, modulation of PSGL1 expression is part of the TFH cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation, suggesting these events preceded T-B cell interactions, although they were required for full development of the TFH cell phenotype, including CXCR5 and PD-1 upregulation, and IL-21 synthesis. Bcl6 upregulation and TFH cell differentiation were independent of IL-6 and IL-21, revealing that either cytokine is not absolutely required for development of Bcl6+ TFH cells in vivo. These data increase our understanding of Bcl6 regulation in TFH cells and their differentiation in vivo, and identifies a new surface marker that may be functionally relevant in this subset. PMID:20519643

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Cell suspension cultures of allogenic keratinocytes are efficient carriers for ex vivo gene transfer and accelerate the healing of full-thickness skin wounds by overexpression of human epidermal growth factor.

PubMed

Vranckx, Jan Jeroen; Hoeller, Daniela; Velander, Patrik E M; Theopold, Christoph F P; Petrie, Nicola; Takedo, Akira; Eriksson, Elof; Yao, Feng

2007-01-01

The concept of using growth factor therapy to induce wound repair has been endorsed in studies that show reduced growth factors in wound fluid from chronic and aged wounds. In this study, we used cell suspensions of allogenic keratinocytes as gene-delivery vehicles for human epidermal growth factor (hEGF) and analyzed their impact on wound repair in a porcine wound-healing model. Full-thickness wounds were created on the backs of six Yorkshire pigs and covered with a wound chamber to create a wet wound-healing environment. First, 5 x 10(5) allogenic, autogenic, or mixed keratinocytes were transplanted into wounds and healing parameters were analyzed. Second, we measured long-term reepithelialization and contraction rates from day 8 until day 35. In the third experiment, allogenic keratinocytes were transfected with an hEGF-expressing plasmid pCEP-hEGF and transplanted in full-thickness wounds to improve repair. Wounds treated with autogenic, allogenic, or mixed keratinocytes showed a significantly higher rate of reepithelialization relative to saline-treated control wounds. Repetitive biopsies indicated that the use of allogenic keratinocytes did not lead to long-term wound breakdown. Wounds treated with hEGF-expressing allogenic keratinocytes reepithelialized faster than wounds treated with allogenic keratinocytes or control wounds. With a peak hEGF expression of 920.8 pg/mL, hEGF was detectable until day 5 after transplantation compared with minimal hEGF expression in control wounds. This study shows that allogenic keratinocytes can serve as efficient gene transfer vehicles for ex vivo growth factor delivery to full-thickness wounds and overexpression of hEGF further improves reepithelialization rates.

Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.

PubMed

Zhang, Bin; Liu, Rui; Shi, Dan; Liu, Xingxia; Chen, Yuan; Dou, Xiaowei; Zhu, Xishan; Lu, Chunhua; Liang, Wei; Liao, Lianming; Zenke, Martin; Zhao, Robert C H

2009-01-01

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2-dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

Enhancement or inhibition of PLCγ2 expression in rat hepatocytes by recombinant adenoviral vectors that contain full-length gene or siRNA.

PubMed

Chen, X G; Liu, Y M; Lv, Q X; Ma, J

2017-01-01

We investigated the effects of recombinant adenovirus vectors that overexpress or silence PLCγ2 on the expression of this gene during hepatocyte proliferation. Hepatocytes were isolated, identified by immunofluorescent cytochemical staining and infected by previously constructed Ad-PLCγ2 and Ad-PLCγ2 siRNA1, siRNA2 and siRNA3. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy. Infection percentage was calculated by flow cytometry. mRNA and protein levels of PLCγ2 were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blotting, respectively. The viability of the infected hepatocytes was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We found that nearly 97% of cells were positive for the hepatocyte marker, CK18. After infection of Ad-PLCγ2 and Ad-PLCγ2 siRNA, more than 99% of hepatocytes expressed GFP significantly, and mRNA and protein expression of PLCγ2 was up-regulated significantly in Ad-PLCγ2 infected hepatocytes, but down-regulated in Ad-PLCγ2 siRNA2 infected cells. The cell proliferation rate decreased in PLCγ2-overexpressing cells, while the rate increased in PLCγ2-silencing cells. We verified that recombinant Ad-PLCγ2 and Ad-PLCγ2 siRNA2 were constructed successfully. These two recombinant vectors promoted or decreased the expression of PLCγ2 in rat hepatocytes and affected the cell proliferation rate, which provides a useful tool for further investigation of the role of PLCγ2 in hepatocyte apoptosis.

Isolation and characterization of multipotent human periodontal ligament stem cells.

PubMed

Gay, I C; Chen, S; MacDougall, M

2007-08-01

Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.

Linking loss of sodium-iodide symporter expression to DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyckesvärd, Madeleine Nordén; Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg; Kapoor, Nirmal

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression andmore » transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.« less

Validation of a recombinant human bactericidal/permeability-increasing protein (hBPI) expression vector using murine mammary gland tumor cells and the early development of hBPI transgenic goat embryos.

PubMed

Gui, Tao; Liu, Xing; Tao, Jia; Chen, Jianwen; Li, Yunsheng; Zhang, Meiling; Wu, Ronghua; Zhang, Yuanliang; Peng, Kaisong; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2013-12-01

Human bactericidal/permeability-increasing protein (hBPI) is the only antibacterial peptide which acts against both gram-negative bacteria and neutralizes endotoxins in human polymorphonuclear neutrophils; therefore, hBPI is of great value in clinical applications. In the study, we constructed a hBPI expression vector (pBC1-Loxp-Neo-Loxp-hBPI) containing the full-length hBPI coding sequence which could be specifically expressed in the mammary gland. To validate the function of the vector, in vitro cultured C127 (mouse mammary Carcinoma Cells) were transfected with the vector, and the transgenic cell clones were selected to express hBPI by hormone induction. The mRNA and protein expression of hBPI showed that the constructed vector was effective and suitable for future application in producing mammary gland bioreactor. Then, female and male goat fibroblasts were transfected with the vector, and two male and two female transgenic clonal cell lines were obtained. Using the transgenic cell lines as nuclear donors for somatic cell nuclear transfer, the reconstructed goat embryos produced from all four clones could develop to blastocysts in vitro. In conclusion, we constructed and validated an efficient mammary gland-specific hBPI expression vector, pBC1-Loxp-Neo-Loxp-hBPI, and transgenic hBPI goat embryos were successfully produced, laying foundations for future production of recombinant hBPI in goat mammary gland. Copyright © 2013 Elsevier B.V. All rights reserved.

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

PubMed

Ryan, G R; Dai, X M; Dominguez, M G; Tong, W; Chuan, F; Chisholm, O; Russell, R G; Pollard, J W; Stanley, E R

2001-07-01

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

PubMed Central

Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

2016-01-01

RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB’s function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

PubMed

Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

2016-05-01

RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

PubMed Central

Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.; Heck, Diane E.; Gerecke, Donald R.; Sinko, Patrick J.; Laskin, Debra L.; Laskin, Jeffrey D.

2010-01-01

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100–1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300–1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE2 synthases, leukotriene (LT) A4 hydrolase and LTC4 synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1–2 (GSTA1–2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics. PMID:20840853

Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide.

PubMed

Black, Adrienne T; Hayden, Patrick J; Casillas, Robert P; Heck, Diane E; Gerecke, Donald R; Sinko, Patrick J; Laskin, Debra L; Laskin, Jeffrey D

2010-12-01

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics. Copyright © 2010 Elsevier Inc. All rights reserved.

Signal Transducer and Activator of Transcription 1 Plays a Pivotal Role in RET/PTC3 Oncogene-induced Expression of Indoleamine 2,3-Dioxygenase 1.

PubMed

Moretti, Sonia; Menicali, Elisa; Nucci, Nicole; Voce, Pasquale; Colella, Renato; Melillo, Rosa Marina; Liotti, Federica; Morelli, Silvia; Fallarino, Francesca; Macchiarulo, Antonio; Santoro, Massimo; Avenia, Nicola; Puxeddu, Efisio

2017-02-03

Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it exerts an immunosuppressive function as part of an acquired mechanism of immune escape. Recently, we demonstrated that IDO1 expression is significantly higher in all thyroid cancer histotypes compared with normal thyroid and that its expression levels correlate with T regulatory (Treg) lymphocyte densities in the tumor microenvironment. BRAF V600E - and RET/PTC3-expressing PcCL3 cells were used as cellular models for the evaluation of IDO1 expression in thyroid carcinoma cells and for the study of involved signal transduction pathways. BRAF V600E -expressing PcCL3 cells did not show IDO1 expression. Conversely, RET/PTC3-expressing cells were characterized by a high IDO1 expression. Moreover, we found that, the STAT1-IRF1 pathway was instrumental for IDO1 expression in RET/PTC3 expressing cells. In detail, RET/PTC3 induced STAT1 overexpression and phosphorylation at Ser-727 and Tyr-701. STAT1 transcriptional regulation appeared to require activation of the canonical NF-κB pathway. Conversely, activation of the MAPK and PI3K-AKT pathways primarily regulated Ser-727 phosphorylation, whereas a physical interaction between RET/PTC3 and STAT1, followed by a direct tyrosine phosphorylation event, was necessary for STAT1 Tyr-701 phosphorylation. These data provide the first evidence of a direct link between IDO1 expression and the oncogenic activation of RET in thyroid carcinoma and describe the involved signal transduction pathways. Moreover, they suggest possible novel molecular targets for the abrogation of tumor microenvironment immunosuppression. The detection of those targets is becoming increasingly important to yield the full function of novel immune checkpoint inhibitors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seong K., E-mail: skim1@lsuhsc.edu; Kim, Seongman; Dai Gan

2011-09-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% ofmore » control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: > We examine the functional domains of IR2P that mediates negative regulation. > IR2P inhibits at the transcriptional level. > DNA-binding mutant or TFIIB-binding mutant fails to inhibit. > C-terminal aa 707 to 1116 are required for full inhibition. > Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.« less

Progressive lentivirus infection induces natural killer cell receptor-expressing B cells in the gastrointestinal tract.

PubMed

Manickam, Cordelia; Nwanze, Chiadika; Ram, Daniel R; Shah, Spandan V; Smith, Scott; Jones, Rhianna; Hueber, Brady; Kroll, Kyle; Varner, Valerie; Goepfert, Paul; Jost, Stephanie; Reeves, R Keith

2018-05-03

Recently, a seemingly novel innate immune cell subset bearing features of natural killer and B cells was identified in mice. So-called NKB cells appear as first responders to infections, but whether this cell population is truly novel or is in fact a subpopulation of B cells and exists in higher primates remains unclear. The objective of this study was to identify NKB cells in primates and study the impact of HIV/SIV infections. NKB cells were quantified in both naïve and lentivirus infected rhesus macaques and humans by excluding lineage markers (CD3, CD127), and positive Boolean gating for CD20, NKG2A/C and/or NKp46. Additional phenotypic measures were conducted by RNA-probe and traditional flow cytometry. Circulating cytotoxic NKB cells were found at similar frequencies in humans and rhesus macaques (range, 0.01-0.2% of total lymphocytes). NKB cells were notably enriched in spleen (median, 0.4% of lymphocytes), but were otherwise systemically distributed in tonsil, lymph nodes, colon, and jejunum. Expression of immunoglobulins was highly variable, but heavily favoured IgM and IgA rather than IgG. Interestingly, NKB cell frequencies expanded in PBMC and colon during SIV infection, as did IgG expression, but were generally unaltered in HIV-infected humans. These results suggest a cell type expressing both natural killer and B-cell features exists in rhesus macaques and humans and are perturbed by HIV/SIV infection. The full functional niche remains unknown, but the unique phenotype and systemic distribution could make NKB cells unique targets for immunotherapeutics or vaccine strategies.

Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

PubMed

Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

2010-08-06

The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

PubMed

Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

PubMed Central

Bami, Myrto; Episkopou, Vasso; Gavalas, Anthony; Gouti, Mina

2011-01-01

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes. PMID:21637844

Rice Shaker Potassium Channel OsKAT1 Confers Tolerance to Salinity Stress on Yeast and Rice Cells1[OA

PubMed Central

Obata, Toshihiro; Kitamoto, Hiroko K.; Nakamura, Atsuko; Fukuda, Atsunori; Tanaka, Yoshiyuki

2007-01-01

We screened a rice (Oryza sativa L. ‘Nipponbare’) full-length cDNA expression library through functional complementation in yeast (Saccharomyces cerevisiae) to find novel cation transporters involved in salt tolerance. We found that expression of a cDNA clone, encoding the rice homolog of Shaker family K+ channel KAT1 (OsKAT1), suppressed the salt-sensitive phenotype of yeast strain G19 (Δena1–4), which lacks a major component of Na+ efflux. It also suppressed a K+-transport-defective phenotype of yeast strain CY162 (Δtrk1Δtrk2), suggesting the enhancement of K+ uptake by OsKAT1. By the expression of OsKAT1, the K+ contents of salt-stressed G19 cells increased during the exponential growth phase. At the linear phase, however, OsKAT1-expressing G19 cells accumulated less Na+ than nonexpressing cells, but almost the same K+. The cellular Na+ to K+ ratio of OsKAT1-expressing G19 cells remained lower than nonexpressing cells under saline conditions. Rice cells overexpressing OsKAT1 also showed enhanced salt tolerance and increased cellular K+ content. These functions of OsKAT1 are likely to be common among Shaker K+ channels because OsAKT1 and Arabidopsis (Arabidopsis thaliana) KAT1 were able to complement the salt-sensitive phenotype of G19 as well as OsKAT1. The expression of OsKAT1 was restricted to internodes and rachides of wild-type rice, whereas other Shaker family genes were expressed in various organs. These results suggest that OsKAT1 is involved in salt tolerance of rice in cooperation with other K+ channels by participating in maintenance of cytosolic cation homeostasis during salt stress and thus protects cells from Na+. PMID:17586689

The Lin28 Expression in Stallion Testes.

PubMed

Lee, Geumhui; Jung, Heejun; Yoon, Minjung

2016-01-01

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.

The Lin28 Expression in Stallion Testes

PubMed Central

Yoon, Minjung

2016-01-01

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody. PMID:27798668

Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus.

PubMed

Martínez-Cáceres, E; Jaleco, A C; Res, P; Noteboom, E; Weijer, K; Spits, H

1998-07-01

In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.

RNAi-based glyconanoparticles trigger apoptotic pathways for in vitro and in vivo enhanced cancer-cell killing

NASA Astrophysics Data System (ADS)

Conde, João; Tian, Furong; Hernandez, Yulan; Bao, Chenchen; Baptista, Pedro V.; Cui, Daxiang; Stoeger, Tobias; de La Fuente, Jesus M.

2015-05-01

Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ~80% reduction in tumour size without associated inflammation.Gold glyconanoparticles (GlycoNPs) are full of promise in areas like biomedicine, biotechnology and materials science due to their amazing physical, chemical and biological properties. Here, siRNA GlycoNPs (AuNP@PEG@Glucose@siRNA) in comparison with PEGylated GlycoNPs (AuNP@PEG@Glucose) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo via intratracheal instillation directly into the lungs of B6 albino mice grafted with luciferase-CMT/167 adenocarcinoma cells. siRNA GlycoNPs but not PEGylated GlycoNPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose dependent manner, independent of the inflammatory response, evaluated by bronchoalveolar lavage cell counting. Moreover, in vivo pulmonary delivered siRNA GlycoNPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNAi in tumour tissue, leading to an ~80% reduction in tumour size without associated inflammation. Electronic supplementary information (ESI) available: Synthesis, functionalization and quantification methods for siRNA-gold glyconanoparticles. See DOI: 10.1039/c4nr05742b

Expression characteristics of long noncoding RNA uc.322 and its effects on pancreatic islet function.

PubMed

Zhao, Xiaoqin; Rong, Can; Pan, Fenghui; Xiang, Lizhi; Wang, Xinlei; Hu, Yun

2018-06-28

Increasing evidence indicates that long noncoding RNAs (lncRNAs) perform special biological functions by regulating gene expression through multiple pathways and molecular mechanisms. The aim of this study was to explore the expression characteristics of lncRNA uc.322 in pancreatic islet cells and its effects on the secretion function of islet cells. Bioinformatics analysis was used to detect the lncRNA uc.322 sequence, location, and structural features. Expression of lncRNA uc.322 in different tissues was detected by quantitative polymerase chain reaction analyses. Quantitative polymerase chain reaction, Western blot analysis, adenosine triphosphate determination, glucose-stimulated insulin secretion, and enzyme-linked immunosorbent assay were used to evaluate the effects of lncRNA uc.322 on insulin secretion. The results showed that the full-length of lncRNA uc.322 is 224 bp and that it is highly conserved in various species. Bioinformatics analysis revealed that lncRNA uc.322 is located on chr7:122893196-122893419 (GRCH37/hg19) within the SRY-related HMG-box 6 gene exon region. Compared with other tissues, lncRNA uc.322 is highly expressed in pancreatic tissue. Upregulation of lncRNA uc.322 expression increases the insulin transcription factors pancreatic and duodenal homeobox 1 and Forkhead box O1 expression, promotes insulin secretion in the extracellular fluid of Min6 cells, and increases the adenosine triphosphate concentration. On the other hand, knockdown of lncRNA uc.322 has opposite effects on Min6 cells. Overall, this study showed that upregulation of lncRNA uc.322 in islet β-cells can increase the expression of insulin transcription factors and promote insulin secretion, and it may be a new therapeutic target for diabetes. © 2018 Wiley Periodicals, Inc.

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

PubMed

Pfisterer, P; König, H; Hess, J; Lipowsky, G; Haendler, B; Schleuning, W D; Wirth, T

1996-11-01

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

MACF1 Overexpression by Transfecting the 21 kbp Large Plasmid PEGFP-C1A-ACF7 Promotes Osteoblast Differentiation and Bone Formation.

PubMed

Zhang, Yan; Yin, Chong; Hu, Lifang; Chen, Zhihao; Zhao, Fan; Li, Dijie; Ma, Jianhua; Ma, Xiaoli; Su, Peihong; Qiu, Wuxia; Yang, Chaofei; Wang, Pai; Li, Siyu; Zhang, Ge; Wang, Liping; Qian, Airong; Xian, Cory J

2018-02-01

Microtubule actin crosslinking factor 1 (MACF1) is a large spectraplakin protein known to have crucial roles in regulating cytoskeletal dynamics, cell migration, growth, and differentiation. However, its role and action mechanism in bone remain unclear. The present study investigated optimal conditions for effective transfection of the large plasmid PEGFP-C1A-ACF7 (∼21 kbp) containing full-length human MACF1 cDNA, as well as the potential role of MACF1 in bone formation. To enhance MACF1 expression, the plasmid was transfected into osteogenic cells by electroporation in vitro and into mouse calvaria with nanoparticles. Then, transfection efficiency, osteogenic marker expression, calvarial thickness, and bone formation were analyzed. Notably, MACF1 overexpression triggered a drastic increase in osteogenic gene expression, alkaline phosphatase activity, and matrix mineralization in vitro. Mouse calvarial thickness, mineral apposition rate, and osteogenic marker protein expression were significantly enhanced by local transfection. In addition, MACF1 overexpression promoted β-catenin expression and signaling. In conclusion, MACF1 overexpression by transfecting the large plasmid containing full-length MACF1 cDNA promotes osteoblast differentiation and bone formation via β-catenin signaling. Current data will provide useful experimental parameters for the transfection of large plasmids and a novel strategy based on promoting bone formation for prevention and therapy of bone disorders.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

PubMed

Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

2011-12-01

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

Immunolocalization of osteopontin in dysplasias and squamous cell carcinomas arising from oral epithelium.

PubMed

Aravind, Thara; Janardhanan, Mahija; Rakesh, S; Savithri, Vindhya; Unnikrishnan, U G

2017-01-01

Early detection of oral squamous cell carcinoma (OSCC) remains one of the most efficient ways to ensure patient survival and improved quality of life. Although specific biomarkers related to OSCC have been investigated, a useful biomarker that assesses the transition potential of potentially malignant lesion to OSCC remains to be found. Osteopontin (OPN) has been recognized as an important factor in tumorigenesis and their expression in OSCC have been investigated earlier. In the present study, evaluation of OPN expression in premalignant and malignant lesions has been carried out to assess their possible role as a biomarker in the early diagnosis and prognosis of OSCC. The objective of this study is to evaluate the role of OPN as a biomarker in the diagnosis and prognosis of OSCC. The study group consisted of archival paraffin-embedded blocks of ten cases each of varying grades of OSCC, oral epithelial dysplasias and epithelial hyperplasias. Sections were subjected to immunohistochemical staining for the biomarker OPN. A positive OPN expression was noticed in epithelial dysplasias and SCC arising from the oral epithelium. A progressive increase in the intensity of staining was seen with increasing grades of dysplasias and a decrease in OPN expression with an increase in grades was observed in OSCC. The expression of OPN in full thickness of epithelium in severe dysplasias, carcinoma in situ, and in the superficial epithelium of OSCC suggest the possibility of considering OPN expression in full epithelial thickness in dysplasias as an indicator for malignant transformation.

Human XPA and XRCC1 DNA repair proteins expressed in yeast, Saccharomyces cerevisiae.

PubMed

Pushnova, E A; Ostanin, K; Thelen, M P

2001-11-01

Human XPA and XRCC1 DNA repair proteins have been expressed in a series of novel yeast episomal vectors. Expression of XPA cDNA resulted in synthesis of anti-XPA crossreacting polypeptides of 40 and 42 kDa, the status of the native protein found in human cells. Likewise, the majority of the recombinant XRCC1 found in the yeast intracellular fraction corresponded to the molecular mass of the full-length human protein. Recombinant XPA protein expressed as an NH(2)-terminal polyhistidine fusion could be affinity purified using Ni(2+) agarose. Copyright 2001 Academic Press.

Fruit weight is controlled by Cell Size Regulator encoding a novel protein that is expressed in maturing tomato fruits

PubMed Central

Chakrabarti, Manohar; Liu, Xiaoxi; Wang, Yanping; Ramos, Alexis

2017-01-01

Increases in fruit weight of cultivated vegetables and fruits accompanied the domestication of these crops. Here we report on the positional cloning of a quantitative trait locus (QTL) controlling fruit weight in tomato. The derived allele of Cell Size Regulator (CSR-D) increases fruit weight predominantly through enlargement of the pericarp areas. The expanded pericarp tissues result from increased mesocarp cell size and not from increased number of cell layers. The effect of CSR on fruit weight and cell size is found across different genetic backgrounds implying a consistent impact of the locus on the trait. In fruits, CSR expression is undetectable early in development from floral meristems to the rapid cell proliferation stage after anthesis. Expression is low but detectable in growing fruit tissues and in or around vascular bundles coinciding with the cell enlargement stage of the fruit maturation process. CSR encodes an uncharacterized protein whose clade has expanded in the Solanaceae family. The mutant allele is predicted to encode a shorter protein due to a 1.4 kb deletion resulting in a 194 amino-acid truncation. Co-expression analyses and GO term enrichment analyses suggest association of CSR with cell differentiation in fruit tissues and vascular bundles. The derived allele arose in Solanum lycopersicum var cerasiforme and appears completely fixed in many cultivated tomato’s market classes. This finding suggests that the selection of this allele was critical to the full domestication of tomato from its intermediate ancestors. PMID:28817560

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

“Inclonals”

PubMed Central

Hakim, Rahely

2009-01-01

Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, high yields (up to 50 mg/L of shake flask culture) of highly purified (>90%) full-length antibodies and antibody-toxin fusions were obtained. The bacterially produced antibodies, named “Inclonals,” equaled the performance of the same IgGs that were produced using conventional mammalian cell culture in binding properties as well as in cell killing potency. The rapid and cost effective IgG production process and the high quality of the resultant product may make the bacterial production of full-length IgG and IgG-drug fusion proteins an attractive option for antibody production and a significant contribution to recombinant antibody technology. PMID:20065645

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells.

PubMed

Weber, Marlen; Apostolova, Galina; Widera, Darius; Mittelbronn, Michel; Dechant, Georg; Kaltschmidt, Barbara; Rohrer, Hermann

2015-02-01

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage. © 2014 AlphaMed Press.

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

PubMed

Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim

2005-11-01

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Lamin B receptor regulates the growth and maturation of myeloid progenitors via its sterol reductase domain: implications for cholesterol biosynthesis in regulating myelopoiesis.

PubMed

Subramanian, Gayathri; Chaudhury, Pulkit; Malu, Krishnakumar; Fowler, Samantha; Manmode, Rahul; Gotur, Deepali; Zwerger, Monika; Ryan, David; Roberti, Rita; Gaines, Peter

2012-01-01

Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin-binding domains plus a C-terminal sterol Δ(14) reductase domain. LBR expression increases during neutrophil differentiation, and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus, LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (erythroid, myeloid, and lymphoid [EML]-derived promyelocytes) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. In this study, we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EML-derived promyelocytes, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full-length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ(14) reductase domain of LBR plays a critical role in cholesterol biosynthesis and that this process is essential to both myeloid cell growth and functional maturation.

Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2.

PubMed

Mavri-Damelin, Demetra; Eaton, Simon; Damelin, Leonard H; Rees, Myrddin; Hodgson, Humphrey J F; Selden, Clare

2007-01-01

A possible cell source for a bio-artificial liver is the human hepatblastoma-derived cell line HepG2 as it confers many hepatocyte functions, however, the urea cycle is not maintained resulting in the lack of ammonia detoxification via this cycle. We investigated urea cycle activity in HepG2 cells at both a molecular and biochemical level to determine the causes for the lack of urea cycle expression, and subsequently addressed reinstatement of the cycle by gene transfer. Metabolic labelling studies showed that urea production from 15N-ammonium chloride was not detectable in HepG2 conditioned medium, nor could 14C-labelled urea cycle intermediates be detected. Gene expression data from HepG2 cells revealed that although expression of three urea cycle genes Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase was evident, Ornithine Transcarbamylase and Arginase I expression were completely absent. These results were confirmed by Western blot for arginase I, where no protein was detected. Radiolabelled enzyme assays showed that Ornithine Transcarbamylase functional activity was missing but that Carbamoyl Phosphate Synthase I, Arginosuccinate Synthetase and Arginosuccinate Lyase were functionally expressed at levels comparable to cultured primary human hepatocytes. To restore the urea cycle, HepG2 cells were transfected with full length Ornithine Transcarbamylase and Arginase I cDNA constructs under a CMV promoter. Co-transfected HepG2 cells displayed complete urea cycle activity, producing both labelled urea and urea cycle intermediates. This strategy could provide a cell source capable of urea synthesis, and hence ammonia detoxificatory function, which would be useful in a bio-artificial liver.

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.

2007-06-29

Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells,more » not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.« less

LDL oxidation by THP-1 monocytes: implication of HNP-1, SgIII and DMT-1.

PubMed

He, Chunyan; Huang, Rui; Du, Fen; Zheng, Fang; Wei, Lei; Wu, Junzhu

2009-04-01

Oxidized low-density lipoprotein (oxLDL) plays an important role in the pathogenesis of atherosclerosis. However, the mechanisms of the initiation and progression of LDL oxidation by cells are still unknown. We investigated the molecular mechanism underlying THP-1 cell-mediated LDL oxidation. LDL oxidation was monitored at 234 nm by detecting the formation of conjugated dienes. cDNA array analysis was applied to profile changes in gene expression of human THP-1 monocytes in response to LDL stimulation. The mRNA and protein levels of secretogranin III (SgIII), divalent metal transporter (DMT-1) and human alpha-defensin 1 (HNP-1) were determined by real-time RT-PCR and Western blotting respectively. Eukaryotic expression vectors containing full-length cDNA sequence of HNP-1 (pEGFP-C1/HNP-1) SgIII (pEGFP-C1/SgIII) or DMT-1 (pEGFP-C1/DMT-1) were constructed and transfected to THP-1 cells. The effects of overexpression of these three genes on THP-1 cell-mediated LDL oxidation were observed. LDL oxidation was most pronounced after LDL was incubated with THP-1 cells for 9 h. 1651 genes in total were detected by cDNA array analysis in THP-1 cells with or without LDL treatment for 9 h. Thirteen genes with >2-fold relative expression difference were identified, including nine genes whose expression was up-regulated and four genes whose expression was down-regulated. Among the up-regulated genes, SgIII, DMT-1 and HNP-1 were reported to be associated with atherosclerosis. The increased mRNA expressions of these three genes were confirmed by real-time RT-PCR. Western blotting analysis demonstrated that protein expressions of SgIII and DMT-1 were also enhanced in THP-1 cells in response to LDL. Furthermore, transient overexpression of HNP-1, SgIII or DMT-1 in THP-1 cells significantly increased THP-1 cell-mediated LDL oxidation. Our data suggest that SgIII, DMT-1 and HNP-1 are implicated in cell-mediated LDL oxidation.

Molecular identification and expression analysis of a natural killer cell enhancing factor (NKEF) from rock bream Oplegnathus fasciatus and the biological activity of its recombinant protein

PubMed Central

Kim, Ju-Won; Choi, Hye-Sung; Kwon, Mun-Gyeong; Park, Myoung-Ae; Hwang, Jee-Youn; Kim, Do-Hyung; Park, Chan-Il

2011-01-01

Natural killer cell enhancing factor (NKEF) belongs to the defined peroxiredoxin (Prx) family. Rock bream NKEF cDNA was identified by expressed sequence tag (EST) analysis of rock bream liver that was stimulated with the LPS. The full-length RbNKEF cDNA (1062 bp) contained an open reading frame (ORF) of 594 bp encoding 198 amino acids. RbNKEF was significantly expressed in the gill, liver, and intestine. mRNA expression of NKEF in the head kidney was examined under viral and bacterial challenge via real-time RT-PCR. Experimental challenge of rock bream with Edwardsiella tarda, Streptococcus iniae, and RSIV resulted in significant increases in RbNKEF mRNA in the head kidney. To obtain a recombinant NKEF, the RbNKEF ORF was expressed in Escherichia coli BL21 (DE3), and the purified soluble protein exhibited a single band corresponding to the predicted molecular mass. When kidney leucocytes were treated with a high concentration of rRbNKEF (10 μg/mL), they exhibited significantly enhanced cell proliferation and viability under oxidative stress. PMID:24371552

Recombinant Domain V of Human Perlecan Is a Bioactive Vascular Proteoglycan.

PubMed

Rnjak-Kovacina, Jelena; Tang, Fengying; Lin, Xiaoting; Whitelock, John M; Lord, Megan S

2017-12-01

The C-terminal domain V of the extracellular matrix proteoglycan perlecan plays unique and often divergent roles in a number of biological processes, including angiogenesis, vascular cell interactions, wound healing, and autophagy. Recombinant forms of domain V have been proposed as therapeutic agents for the treatment of cancer, stroke, and the development of cardiovascular devices and bioartificial tissues. However, the effect of domain V appears to be related to the differences in domain V structure and function observed in different expression systems and environments and exactly how this occurs is not well understood. In this study, the sequence from amino acid 3626 to 4391 of the perlecan protein core, which includes domain V, is expressed in HEK-293 cells and purified as a secreted product from conditioned media. This recombinant domain V (rDV) is expressed as a proteoglycan decorated with heparan sulfate and chondroitin sulfate chains and supports endothelial cell interactions to the same extent as full-length perlecan. This expression system serves as an important model of recombinant proteoglycan expression, as well as a source of biologically active rDV for therapeutic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis.

PubMed

Hernández-Chirlaque, Cristina; Gámez-Belmonte, Reyes; Ocón, Borja; Martínez-Moya, Patricia; Wirtz, Stefan; Sánchez de Medina, Fermín; Martínez-Augustin, Olga

2017-07-01

Two alkaline phosphatase isoforms, intestinal [IAP] and tissue non-specific alkaline phosphatase [TNAP], are coexpressed in mouse colon, with the latter predominating in colitis. We aimed to examine the role of TNAP in T lymphocytes, using heterozygous TNAP+/- mice [as TNAP-/- mice are non-viable]. In vitro primary cultures and in vivo T cell models using TNAP+/- mice were used. Stimulated splenocytes [lipopolysaccharide and concanavalin A] and T lymphocytes [concanavalin A and a-CD3/a-CD28] showed a decreased cytokine production and expression when compared with wild-type [WT] cells. Decreased T cell activation was reproduced by the TNAP inhibitors levamisole, theophylline, and phenylalanine in WT cells. Intraperitoneal administration of anti-CD3 in vivo resulted in reduced plasma cytokine levels, and decreased activation of splenocytes and T cells ex vivo in TNAP+/- mice. We further tested the hypothesis that TNAP expressed in T lymphocytes is involved in T cell activation and inflammation, using the lymphocyte transfer model of colitis. Rag1-/- mice were transferred with T naïve cells [CD4+ CD62L+] from TNAP+/- or WT mice and developed colitis, which was attenuated in the group receiving TNAP+/- cells. Compared with WT, T cells from TNAP+/- mice showed a decreased capacity for proliferation, with no change in differentiation. Our results offer clear evidence that TNAP modulates T lymphocyte function and specifically T cell-dependent colitis. This was associated with distinct changes in the type of TNAP expressed, probably because of changes in glycosylation. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

Expression and purification of functional PDGF receptor beta.

PubMed

Shang, Qingbin; Zhao, Liang; Wang, Xiaojing; Wang, Meimei; Sui, Sen-Fang; Mi, Li-Zhi

2017-07-29

Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved. It is caused, at least in part, by the technical challenges in the expression and purification of the functional, full-length PDGF receptors. Herein, we reported our experimental details in expression and purification of the full-length PDGFRβ from mammalian cells. We found that purified PDGFRβ remained in two different oligomeric states, presumably the monomer and the dimer, with basal kinase activity in detergent micelles. Addition of PDGF-B promoted dimerization and elevated kinase activity of the receptor, suggesting that purified receptors were functional. Copyright © 2017 Elsevier Inc. All rights reserved.

Integrated Quantitative Transcriptome Maps of Human Trisomy 21 Tissues and Cells

PubMed Central

Pelleri, Maria Chiara; Cattani, Chiara; Vitale, Lorenza; Antonaros, Francesca; Strippoli, Pierluigi; Locatelli, Chiara; Cocchi, Guido; Piovesan, Allison; Caracausi, Maria

2018-01-01

Down syndrome (DS) is due to the presence of an extra full or partial chromosome 21 (Hsa21). The identification of genes contributing to DS pathogenesis could be the key to any rational therapy of the associated intellectual disability. We aim at generating quantitative transcriptome maps in DS integrating all gene expression profile datasets available for any cell type or tissue, to obtain a complete model of the transcriptome in terms of both expression values for each gene and segmental trend of gene expression along each chromosome. We used the TRAM (Transcriptome Mapper) software for this meta-analysis, comparing transcript expression levels and profiles between DS and normal brain, lymphoblastoid cell lines, blood cells, fibroblasts, thymus and induced pluripotent stem cells, respectively. TRAM combined, normalized, and integrated datasets from different sources and across diverse experimental platforms. The main output was a linear expression value that may be used as a reference for each of up to 37,181 mapped transcripts analyzed, related to both known genes and expression sequence tag (EST) clusters. An independent example in vitro validation of fibroblast transcriptome map data was performed through “Real-Time” reverse transcription polymerase chain reaction showing an excellent correlation coefficient (r = 0.93, p < 0.0001) with data obtained in silico. The availability of linear expression values for each gene allowed the testing of the gene dosage hypothesis of the expected 3:2 DS/normal ratio for Hsa21 as well as other human genes in DS, in addition to listing genes differentially expressed with statistical significance. Although a fraction of Hsa21 genes escapes dosage effects, Hsa21 genes are selectively over-expressed in DS samples compared to genes from other chromosomes, reflecting a decisive role in the pathogenesis of the syndrome. Finally, the analysis of chromosomal segments reveals a high prevalence of Hsa21 over-expressed segments over the other genomic regions, suggesting, in particular, a specific region on Hsa21 that appears to be frequently over-expressed (21q22). Our complete datasets are released as a new framework to investigate transcription in DS for individual genes as well as chromosomal segments in different cell types and tissues. PMID:29740474

Expression of Truncated Neurokinin-1 Receptor in Childhood Neuroblastoma is Independent of Tumor Biology and Stage.

PubMed

Pohl, Alexandra; Kappler, Roland; Mühling, Jakob; VON Schweinitz, Dietrich; Berger, Michael

2017-11-01

Neuroblastoma is an embryonal malignancy arising from the aberrant growth of neural crest progenitor cells of the sympathetic nervous system. The tachykinin receptor 1 (TACR1) - substance P complex is associated with tumoral angiogenesis and cell proliferation in a variety of cancer types. Inhibition of TACR1 was recently described to impede growth of NB cell lines. However, the relevance of TACR1 in clinical settings is unknown. We investigated gene expression levels of full-length and truncated TACR1 in 59 neuroblastomas and correlated these data with the patients' clinical parameters such as outcome, metastasis, International Neuroblastoma Staging System (INSS) status, MYCN proto-oncogene, bHLH transcription factor (MYCN) status, gender and age. Our results indicated that TACR1 is ubiquitously expressed in neuroblastoma but expression levels are independent of clinical parameters. Our data suggest that TACR1 might serve as a potent anticancer target in a large variety of patients with neuroblastoma, independent of tumor biology and clinical stage. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

PubMed

Sethi, Isha; Romano, Rose-Anne; Gluck, Christian; Smalley, Kirsten; Vojtesek, Borivoj; Buck, Michael J; Sinha, Satrajit

2015-08-07

The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

RNA interference of pax2 inhibits growth of transplanted human endometrial cancer cells in nude mice

PubMed Central

Zhang, Li-Ping; Shi, Xiao-Yan; Zhao, Chang-Yin; Liu, Yong-Zhen; Cheng, Ping

2011-01-01

The development of human endometrial carcinoma (HEC) is a complex pathologic process involves several oncogenes and tumor suppressor genes. The full-length paired-box gene 2 (pax2), a recently discovered Oncogene, promotes cell proliferation and growth and inhibits apoptosis of HEC cells. Here, we examined the effect of pax2 small interfering RNA (siRNA) on the growth of transplanted HEC cells in nude mice. The expression of Pax2 in 21 cases of normal endometrium and 38 cases of HEC was examined by immohistochemistry (IHC). HEC models were developed by subcutaneously transferring HEC cells into nude mice, followed by treatment with empty lentivirus vector, lentivirus vector-based pax2 siRNA, and phosphate buffered saline, respectively. Four weeks later, tumor size was measured, tumor inhibition rate was calculated, and histological analyses were conducted after staining with hematoxylin and eosin. The expression of Pax2 and Bcl-2 was detected by Western blot; proliferating cell nuclear antigen (PCNA) was detected by IHC. Significant differences were observed in the positive rate of Pax2 between normal endometrium and HEC (14.2% vs. 60.5%, P < 0.01). The expression index of Pax2 in well differentiated tumors was 1.88 ± 1.68, much lower than that in tumors of moderate (3.07 ± 1.96, P < 0.05) or poor differentiation (5.45 ± 2.76, P < 0.01). Tumor necrosis increased, nuclear basophilia stain decreased, tumor growth was inhibited, and PCNA, Pax2, and Bcl-2 expression was reduced in HEC models treated with pax2 siRNA. These results indicate that Pax2 expression is related to HEC tumor biology with the increased expression of Pax2 correlated to malignancy. pax2 siRNA down-regulates Pax2 expression and inhibits tumorigenesis of HEC in nude mice, possibly due to cell apoptosis and the inhibition of tumor proliferation induced by down-regulation of Bcl-2. PMID:21627862

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

PubMed

Wang, Guang; Chen, En-Ni; Liang, Chang; Liang, Jianxin; Gao, Lin-Rui; Chuai, Manli; Münsterberg, Andrea; Bao, Yongping; Cao, Liu; Yang, Xuesong

2018-04-01

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 + neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 + and HNK-1 + cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 + neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU + neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

PubMed Central

Reedijk, M; Liu, X Q; Pawson, T

1990-01-01

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice.

PubMed

Klingenberg, Jennifer M; McFarland, Kevin L; Friedman, Aaron J; Boyce, Steven T; Aronow, Bruce J; Supp, Dorothy M

2010-02-01

Bioengineered skin substitutes can facilitate wound closure in severely burned patients, but deficiencies limit their outcomes compared with native skin autografts. To identify gene programs associated with their in vivo capabilities and limitations, we extended previous gene expression profile analyses to now compare engineered skin after in vivo grafting with both in vitro maturation and normal human skin. Cultured skin substitutes were grafted on full-thickness wounds in athymic mice, and biopsy samples for microarray analyses were collected at multiple in vitro and in vivo time points. Over 10,000 transcripts exhibited large-scale expression pattern differences during in vitro and in vivo maturation. Using hierarchical clustering, 11 different expression profile clusters were partitioned on the basis of differential sample type and temporal stage-specific activation or repression. Analyses show that the wound environment exerts a massive influence on gene expression in skin substitutes. For example, in vivo-healed skin substitutes gained the expression of many native skin-expressed genes, including those associated with epidermal barrier and multiple categories of cell-cell and cell-basement membrane adhesion. In contrast, immunological, trichogenic, and endothelial gene programs were largely lacking. These analyses suggest important areas for guiding further improvement of engineered skin for both increased homology with native skin and enhanced wound healing.

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

PubMed Central

Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

1988-01-01

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

Hypoxia and prostaglandin E receptor 4 signalling pathways synergise to promote endometrial adenocarcinoma cell proliferation and tumour growth.

PubMed

Catalano, Rob D; Wilson, Martin R; Boddy, Sheila C; McKinlay, Andrew T M; Sales, Kurt J; Jabbour, Henry N

2011-05-12

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E(2). PTGS2 expression and PGE(2) biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE(2) regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1-4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE(2) and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE(2) and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells.

Hypoxia and Prostaglandin E Receptor 4 Signalling Pathways Synergise to Promote Endometrial Adenocarcinoma Cell Proliferation and Tumour Growth

PubMed Central

Catalano, Rob D.; Wilson, Martin R.; Boddy, Sheila C.; McKinlay, Andrew T. M.; Sales, Kurt J.; Jabbour, Henry N.

2011-01-01

The prostaglandin endoperoxide synthase (PTGS) pathway is a potent driver of tumour development in humans by enhancing the biosynthesis and signalling of prostaglandin (PG) E2. PTGS2 expression and PGE2 biosynthesis is elevated in endometrial adenocarcinoma, however the mechanism whereby PTGS and PGE2 regulate endometrial tumour growth is unknown. Here we investigated (a) the expression profile of the PGE synthase enzymes (PTGES, PTGES-2, PTGES-3) and PGE receptors (PTGER1–4) in endometrial adenocarcinomas compared with normal endometrium and (b) the role of PTGER4 in endometrial tumorigenesis in vivo. We found elevated expression of PTGES2 and PTGER4 and suppression of PTGER1 and PTGER3 in endometrial adenocarcinomas compared with normal endometrium. Using WT Ishikawa endometrial adenocarcinoma cells and Ishikawa cells stably transfected with the full length PTGER4 cDNA (PTGER4 cells) xenografted in the dorsal flanks of nude mice, we show that PTGER4 rapidly and significantly enhances tumour growth rate. Coincident with enhanced PTGER4-mediated tumour growth we found elevated expression of PTGS2 in PTGER4 xenografts compared with WT xenografts. Furthermore we found that the augmented growth rate of the PTGER4 xenografts was not due to enhanced angiogenesis, but regulated by an increased proliferation index and hypoxia. In vitro, we found that PGE2 and hypoxia independently induce expression of PTGER4 indicating two independent pathways regulating prostanoid receptor expression. Finally we have shown that PGE2 and hypoxia synergise to promote cellular proliferation of endometrial adenocarcinoma cells. PMID:21589857

Selective silencing of full-length CD80 but not IgV-CD80 leads to impaired clonal deletion of self-reactive T cells and altered regulation of immune responses.

PubMed

Bugeon, L; Hargreaves, R E; Crompton, T; Outram, S; Rahemtulla, A; Porter, A C; Dallman, M J

2001-01-01

Co-stimulation provided by the B7 family of proteins underpins the development of protective immunity. There are three identified members of this family: CD80, its splice variant IgV-CD80 and CD86. It has hitherto been difficult to analyze the expression and function of IgV-CD80 since there are no appropriate reagents capable of distinguishing it from CD80. We have generated mice, by gene targeting, the lack CD80 whilst maintaining expression of IgV-CD80. Mutant animals did not delete T cells bearing mammary tumor virus-reactive TCR as efficiently as wild-type animals. We also demonstrate the importance of IgV-CD80 in the responses of recently activated cells and reveal a role for CD80 in sustaining T cell responses. CD86, whilst critical to primary T cell activation, made only a minor contribution to re-activation of normal cells.

Differential Expression of NK Receptors CD94 and NKG2A by T Cells in Rheumatoid Arthritis Patients in Remission Compared to Active Disease

PubMed Central

Walsh, Ceara E.; Ryan, Elizabeth J.; O’Farrelly, Cliona; Golden-Mason, Lucy; FitzGerald, Oliver; Veale, Douglas J.; Bresnihan, Barry; Fearon, Ursula

2011-01-01

Objective TNF inhibitors (TNFi) have revolutionised the treatment of rheumatoid arthritis (RA). Natural killer (NK) cells and Natural Killer Cell Receptor+ T (NKT) cells comprise important effector lymphocytes whose activity is tightly regulated through surface NK receptors (NKRs). Dysregulation of NKRs in patients with autoimmune diseases has been shown, however little is known regarding NKRs expression in patients with TNFi-induced remission and in those who maintain remission vs disease flare following TNFi withdrawal. Methods Patients with RA were recruited for this study, (i) RA patients in clinical remission following a minimum of one year of TNFi therapy (n = −15); (2) Active RA patients, not currently or ever receiving TNFi (n = 18); and healthy control volunteers (n = 15). Patients in remission were divided into two groups: those who were maintained on TNFi and those who withdrew from TNFi and maintained on DMARDS. All patients underwent full clinical assessment. Peripheral blood mononuclear cells were isolated and NKR (CD94, NKG2A, CD161, CD69, CD57, CD158a, CD158b) expression on T-(CD3+CD56−), NK-(CD3−CD56+) and NKT-(CD3+CD56+) cells was determined by flow cytometry. Results Following TNFi withdrawal, percentages and numbers of circulating T cells, NK cells or NKT cell populations were unchanged in patients in remission versus active RA or HCs. Expression of the NKRs CD161, CD57, CD94 and NKG2A was significantly increased on CD3+CD56-T cells from patients in remission compared to active RA (p<0.05). CD3+CD56-T cell expression of CD94 and NKG2A was significantly increased in patients who remained in remission compared with patients whose disease flared (p<0.05), with no differences observed for CD161 and CD57. CD3+CD56− cell expression of NKG2A was inversely related to DAS28 (r = −0.612, p<0.005). Conclusion High CD94/NKG2A expression by T cells was demonstrated in remission patients following TNFi therapy compared to active RA, while low CD94/NKG2A were associated with disease flare following withdrawal of therapy. PMID:22102879

Differential expression of NK receptors CD94 and NKG2A by T cells in rheumatoid arthritis patients in remission compared to active disease.

PubMed

Walsh, Ceara E; Ryan, Elizabeth J; O'Farrelly, Cliona; Golden-Mason, Lucy; FitzGerald, Oliver; Veale, Douglas J; Bresnihan, Barry; Fearon, Ursula

2011-01-01

TNF inhibitors (TNFi) have revolutionised the treatment of rheumatoid arthritis (RA). Natural killer (NK) cells and Natural Killer Cell Receptor+ T (NKT) cells comprise important effector lymphocytes whose activity is tightly regulated through surface NK receptors (NKRs). Dysregulation of NKRs in patients with autoimmune diseases has been shown, however little is known regarding NKRs expression in patients with TNFi-induced remission and in those who maintain remission vs disease flare following TNFi withdrawal. Patients with RA were recruited for this study, (i) RA patients in clinical remission following a minimum of one year of TNFi therapy (n = -15); (2) Active RA patients, not currently or ever receiving TNFi (n = 18); and healthy control volunteers (n = 15). Patients in remission were divided into two groups: those who were maintained on TNFi and those who withdrew from TNFi and maintained on DMARDS. All patients underwent full clinical assessment. Peripheral blood mononuclear cells were isolated and NKR (CD94, NKG2A, CD161, CD69, CD57, CD158a, CD158b) expression on T-(CD3+CD56-), NK-(CD3-CD56+) and NKT-(CD3+CD56+) cells was determined by flow cytometry. Following TNFi withdrawal, percentages and numbers of circulating T cells, NK cells or NKT cell populations were unchanged in patients in remission versus active RA or HCs. Expression of the NKRs CD161, CD57, CD94 and NKG2A was significantly increased on CD3+CD56-T cells from patients in remission compared to active RA (p<0.05). CD3+CD56-T cell expression of CD94 and NKG2A was significantly increased in patients who remained in remission compared with patients whose disease flared (p<0.05), with no differences observed for CD161 and CD57. CD3+CD56- cell expression of NKG2A was inversely related to DAS28 (r = -0.612, p<0.005). High CD94/NKG2A expression by T cells was demonstrated in remission patients following TNFi therapy compared to active RA, while low CD94/NKG2A were associated with disease flare following withdrawal of therapy.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

Minimally Activated CD8 Autoreactive T Cells Specific for IRBP Express a High Level of Foxp3 and Are Functionally Suppressive

PubMed Central

Peng, Yong; Shao, Hui; Ke, Yan; Zhang, Ping; Han, Gencheng; Kaplan, Henry J.; Sun, Deming

2008-01-01

Purpose Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide. Methods Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retin-oid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity. Results CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 μg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-β1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function. Conclusions Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells. PMID:17460277

Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues.

PubMed

Owman, C; Blay, P; Nilsson, C; Lolait, S J

1996-11-12

Using PCR with degenerate primers and screening of a human B-cell lymphoblast cDNA library, a full-length cDNA encoding a 375-amino-acid protein was isolated. It contains seven regions of hydrophobic amino acids probably representing membrane-spanning domains of a novel heptahelix receptor, tentatively named CMKRL2. It shows nearly 30% overall identity with the high-affinity IL8 receptor and similar degree of homology with other chemoattractant receptors, including the "fusin" coreceptors for HIV1. Measurements of various transduction pathways following application of a panel of chemokines to transfected cells failed to evoke any reproducible response. Although the natural ligand for CMKRL2 could, thus, not be identified, receptor expression in spleen and lymph nodes as well as in Burkitt's lymphoma (irrespective of EBV status) supports a functional role in activated B-cells. Receptor message was ubiquitously distributed in normal peripheral tissues and CNS, suggesting that CMKRL2 is expressed in widespread cell populations, such as macrophages and neuroglia.

Hepatitis C Virus-Related Lymphomagenesis in a Mouse Model

PubMed Central

Tsukiyama-Kohara, Kyoko; Sekiguchi, Satoshi; Kasama, Yuri; Salem, Nagla Elwy; Machida, Keigo; Kohara, Michinori

2011-01-01

B cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis C virus (HCV) infection. The mechanism by which HCV infection leads to lymphoproliferative disorder remains unclear. Our group established HCV transgenic mice that expressed the full HCV genome in B cells (RzCD19Cre mice). We observed a 25.0% incidence of diffuse large B cell non-Hodgkin lymphomas (22.2% in male and 29.6% in female mice) within 600 days of birth. Interestingly, RzCD19Cre mice with substantially elevated serum-soluble interleukin-2 receptor α-subunit (sIL-2Rα) levels (>1000 pg/mL) developed B cell lymphomas. Another mouse model of lymphoproliferative disorder was established by persistent expression of HCV structural proteins through disruption of interferon regulatory factor-1 (irf-1_/_/CN2 mice). Irf-1_/_/CN2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders. Moreover, these mice showed increased levels of interleukin (IL)-2, IL-10, and Bcl-2 as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes. PMID:22084693

Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

PubMed Central

Wang, Jin; Gines, Silvia; MacDonald, Marcy E; Gusella, James F

2005-01-01

Background Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 μM, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin. PMID:15649316

HD CAG-correlated gene expression changes support a simple dominant gain of function

PubMed Central

Jacobsen, Jessie C.; Gregory, Gillian C.; Woda, Juliana M.; Thompson, Morgan N.; Coser, Kathryn R.; Murthy, Vidya; Kohane, Isaac S.; Gusella, James F.; Seong, Ihn Sik; MacDonald, Marcy E.; Shioda, Toshi; Lee, Jong-Min

2011-01-01

Huntington's disease is initiated by the expression of a CAG repeat-encoded polyglutamine region in full-length huntingtin, with dominant effects that vary continuously with CAG size. The mechanism could involve a simple gain of function or a more complex gain of function coupled to a loss of function (e.g. dominant negative-graded loss of function). To distinguish these alternatives, we compared genome-wide gene expression changes correlated with CAG size across an allelic series of heterozygous CAG knock-in mouse embryonic stem (ES) cell lines (HdhQ20/7, HdhQ50/7, HdhQ91/7, HdhQ111/7), to genes differentially expressed between Hdhex4/5/ex4/5 huntingtin null and wild-type (HdhQ7/7) parental ES cells. The set of 73 genes whose expression varied continuously with CAG length had minimal overlap with the 754-member huntingtin-null gene set but the two were not completely unconnected. Rather, the 172 CAG length-correlated pathways and 238 huntingtin-null significant pathways clustered into 13 shared categories at the network level. A closer examination of the energy metabolism and the lipid/sterol/lipoprotein metabolism categories revealed that CAG length-correlated genes and huntingtin-null-altered genes either were different members of the same pathways or were in unique, but interconnected pathways. Thus, varying the polyglutamine size in full-length huntingtin produced gene expression changes that were distinct from, but related to, the effects of lack of huntingtin. These findings support a simple gain-of-function mechanism acting through a property of the full-length huntingtin protein and point to CAG-correlative approaches to discover its effects. Moreover, for therapeutic strategies based on huntingtin suppression, our data highlight processes that may be more sensitive to the disease trigger than to decreased huntingtin levels. PMID:21536587

Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies

PubMed Central

Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol

2012-01-01

Summary VP22, encoded by the UL49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. PMID:22569534

A Complex Interaction Between Reduced Reelin Expression and Prenatal Organophosphate Exposure Alters Neuronal Cell Morphology.

PubMed

Mullen, Brian R; Ross, Brennan; Chou, Joan Wang; Khankan, Rana; Khialeeva, Elvira; Bui, Kimberly; Carpenter, Ellen M

2016-06-01

Genetic and environmental factors are both likely to contribute to neurodevelopmental disorders including schizophrenia, autism spectrum disorders, and major depressive disorders. Prior studies from our laboratory and others have demonstrated that the combinatorial effect of two factors-reduced expression of reelin protein and prenatal exposure to the organophosphate pesticide chlorpyrifos oxon-gives rise to acute biochemical effects and to morphological and behavioral phenotypes in adolescent and young adult mice. In the current study, we examine the consequences of these factors on reelin protein expression and neuronal cell morphology in adult mice. While the cell populations that express reelin in the adult brain appear unchanged in location and distribution, the levels of full length and cleaved reelin protein show persistent reductions following prenatal exposure to chlorpyrifos oxon. Cell positioning and organization in the hippocampus and cerebellum are largely normal in animals with either reduced reelin expression or prenatal exposure to chlorpyrifos oxon, but cellular complexity and dendritic spine organization is altered, with a skewed distribution of immature dendritic spines in adult animals. Paradoxically, combinatorial exposure to both factors appears to generate a rescue of the dendritic spine phenotypes, similar to the mitigation of behavioral and morphological changes observed in our prior study. Together, our observations support an interaction between reelin expression and chlorpyrifos oxon exposure that is not simply additive, suggesting a complex interplay between genetic and environmental factors in regulating brain morphology. © The Author(s) 2016.

PAL-12, a new anti-aging hexa-peptoid, inhibits UVB-induced photoaging in human dermal fibroblasts and 3D reconstructed human full skin model, Keraskin-FT™.

PubMed

Song, Daeun; Park, Hyeonji; Lee, Su-Hyon; Kim, Mi Jung; Kim, Eun-Joo; Lim, Kyung-Min

2017-11-01

Peptoids are a class of peptidomimetics whose pharmacological activities are widely investigated owing to their remarkable biological stability. However, the utilities of peptoids as cosmetic functional ingredients have not been fully explored. Here, we investigated anti-aging effects of PAL-12, a new hexa-peptoid, on UVB-induced photoaging in human dermal fibroblasts (HDFs) and a 3D reconstituted human full skin model, Keraskin-FT™. PAL-12 suppressed matrix metalloproteinase-1 (MMP-1) expression induced by UVB irradiation along with the attenuation of MMP-1 secretion as determined by ELISA assay. Interestingly PAL-12 slightly enhanced the expression levels of collagen-1 and fibronectin-1 in HDFs or Keraskin-FT™. In addition, PAL-12 prevented the decrease of cell viability following UVB irradiation. However, PAL-12 failed to affect ROS generation, cell necrosis and apoptosis significantly. Instead, PAL-12 suppressed UVB-induced activation of epidermal growth factor receptors (EGFR), extracellular signal-regulated kinase (ERK) and c-Jun, which may resulted in the attenuation of AP-1-promoted MMP-1 expression. Collectively, these results suggest that PAL-12 might be a novel cosmetic ingredient effective against UVB-induced skin photoaging.

Mutant p53-R273H mediates cancer cell survival and anoikis resistance through AKT-dependent suppression of BCL2-modifying factor (BMF).

PubMed

Tan, B S; Tiong, K H; Choo, H L; Chung, F Fei-Lei; Hii, L-W; Tan, S H; Yap, I K S; Pani, S; Khor, N T W; Wong, S F; Rosli, R; Cheong, S-K; Leong, C-O

2015-07-16

p53 is the most frequently mutated tumor-suppressor gene in human cancers. Unlike other tumor-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the expression of full-length mutant p53 protein. Mutant p53 proteins not only lose their tumor-suppressor function, but may also gain new oncogenic functions and promote tumorigenesis. Here, we showed that silencing of endogenous p53-R273H contact mutant, but not p53-R175H conformational mutant, reduced AKT phosphorylation, induced BCL2-modifying factor (BMF) expression, sensitized BIM dissociation from BCL-XL and induced mitochondria-dependent apoptosis in cancer cells. Importantly, cancer cells harboring endogenous p53-R273H mutant were also found to be inherently resistant to anoikis and lack BMF induction following culture in suspension. Underlying these activities is the ability of p53-R273H mutant to suppress BMF expression that is dependent on constitutively active PI3K/AKT signaling. Collectively, these findings suggest that p53-R273H can specifically drive AKT signaling and suppress BMF expression, resulting in enhanced cell survivability and anoikis resistance. These findings open the possibility that blocking of PI3K/AKT will have therapeutic benefit in mutant p53-R273H expressing cancers.

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

PubMed

Djurisic, S; Teiblum, S; Tolstrup, C K; Christiansen, O B; Hviid, T V F

2015-03-01

The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes.

PubMed

Wagh, Vilas; Pomorski, Alexander; Wilschut, Karlijn J; Piombo, Sebastian; Bernstein, Harold S

2014-06-06

Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

PubMed

Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

2015-01-01

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

PubMed

McIlhinney, R A; Molnár, E

1996-04-01

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

A comparison of scaffold-free and scaffold-based reconstructed human skin models as alternatives to animal use.

PubMed

Kinikoglu, Beste

2017-12-01

Tissue engineered full-thickness human skin substitutes have various applications in the clinic and in the laboratory, such as in the treatment of burns or deep skin defects, and as reconstructed human skin models in the safety testing of drugs and cosmetics and in the fundamental study of skin biology and pathology. So far, different approaches have been proposed for the generation of reconstructed skin, each with its own advantages and disadvantages. Here, the classic tissue engineering approach, based on cell-seeded polymeric scaffolds, is compared with the less-studied cell self-assembly approach, where the cells are coaxed to synthesise their own extracellular matrix (ECM). The resulting full-thickness human skin substitutes were analysed by means of histological and immunohistochemical analyses. It was found that both the scaffold-free and the scaffold-based skin equivalents successfully mimicked the functionality and morphology of native skin, with complete epidermal differentiation (as determined by the expression of filaggrin), the presence of a continuous basement membrane expressing collagen VII, and new ECM deposition by dermal fibroblasts. On the other hand, the scaffold-free model had a thicker epidermis and a significantly higher number of Ki67-positive proliferative cells, indicating a higher capacity for self-renewal, as compared to the scaffold-based model. 2017 FRAME.

Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

PubMed

Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Plasma membrane Ca(2+)ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+)) from the cell, hence regulating Ca(2+) level within cells. Though plant Ca(2+)ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. The 1478 bp promoter sequence of rice plasma membrane Ca(2+)ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+)ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. The rice plasma membrane Ca(2+)ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

Engineered Commensal Bacteria Reprogram Intestinal Cells Into Glucose-Responsive Insulin-Secreting Cells for the Treatment of Diabetes

PubMed Central

Duan, Franklin F.; Liu, Joy H.

2015-01-01

The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1–secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25–33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)–secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo. PMID:25626737

A Fusion Receptor as a Safety Switch, Detection, and Purification Biomarker for Adoptive Transferred T Cells.

PubMed

Wu, Xiuqi; Shi, Bizhi; Zhang, Jiqin; Shi, Zhimin; Di, Shengmeng; Fan, Minliang; Gao, Huiping; Wang, Hai; Gu, Jianren; Jiang, Hua; Li, Zonghai

2017-10-04

The incorporation of an endogenous safety switch represents a rational strategy for the control of toxicities following the administration of adoptive T cell therapies. An ideal safety switch should be capable of depleting the transferred T cells with minimal injury to normal tissues. We generated a fusion receptor by engineering a cryptic 806 epitope of human epidermal growth factor receptor (EGFR) into the N terminus of the full-length human folate receptor 1 (FOLR1), designated as FR806. The expression of FR806 allows transduced T cells to be targeted with CH12, a monoclonal antibody recognizing the 806 epitope, but not wild-type EGFR in healthy tissues. FR806, therefore, constitutes a specific cell-surface marker for the elimination of transduced T cells. We demonstrate that the antibody-drug conjugate (ADC) CH12-MMAF is efficiently internalized by FR806-expressing T cells and has the potential to eliminate them. Transfected T cells could, furthermore, be efficiently detected and purified using CH12 antibodies. In immuno-compromised mice, CH12-MMAF eliminated the majority of transferred T cells expressing FR806 and anti-CD19 chimeric antigen receptor (CAR). The selectivity for the 806 epitope and internalization capacity of FOLR1 makes FR806 an efficient safety switch, which may additionally be used as a detection and purification biomarker for human T cell immunotherapies. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

PubMed

Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

2018-05-15

Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

Immunolocalization of osteopontin in dysplasias and squamous cell carcinomas arising from oral epithelium

PubMed Central

Aravind, Thara; Janardhanan, Mahija; Rakesh, S; Savithri, Vindhya; Unnikrishnan, U G

2017-01-01

Background: Early detection of oral squamous cell carcinoma (OSCC) remains one of the most efficient ways to ensure patient survival and improved quality of life. Although specific biomarkers related to OSCC have been investigated, a useful biomarker that assesses the transition potential of potentially malignant lesion to OSCC remains to be found. Osteopontin (OPN) has been recognized as an important factor in tumorigenesis and their expression in OSCC have been investigated earlier. In the present study, evaluation of OPN expression in premalignant and malignant lesions has been carried out to assess their possible role as a biomarker in the early diagnosis and prognosis of OSCC. Objectives: The objective of this study is to evaluate the role of OPN as a biomarker in the diagnosis and prognosis of OSCC. Materials and Methods: The study group consisted of archival paraffin-embedded blocks of ten cases each of varying grades of OSCC, oral epithelial dysplasias and epithelial hyperplasias. Sections were subjected to immunohistochemical staining for the biomarker OPN. Results: A positive OPN expression was noticed in epithelial dysplasias and SCC arising from the oral epithelium. A progressive increase in the intensity of staining was seen with increasing grades of dysplasias and a decrease in OPN expression with an increase in grades was observed in OSCC. Conclusion: The expression of OPN in full thickness of epithelium in severe dysplasias, carcinoma in situ, and in the superficial epithelium of OSCC suggest the possibility of considering OPN expression in full epithelial thickness in dysplasias as an indicator for malignant transformation. PMID:28479681

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

PubMed

Cappelletti, Pamela; Binda, Elisa; Tunesi, Marta; Albani, Diego; Giordano, Carmen; Molla, Gianluca; Pollegioni, Loredano

2017-10-01

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells

PubMed Central

Alabi, Ola D.; Gunnink, Stephen M.; Kuiper, Benjamin D.; Kerk, Samuel A.; Braun, Emily; Louters, Larry L.

2016-01-01

Aims Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. Main Methods We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Key findings Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 µM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. Significance The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. PMID:24657891

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells.

PubMed

Alabi, Ola D; Gunnink, Stephen M; Kuiper, Benjamin D; Kerk, Samuel A; Braun, Emily; Louters, Larry L

2014-05-02

Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. Copyright © 2014 Elsevier Inc. All rights reserved.

Genetically Engineered Natural Killer Cells as a Means for Adoptive Tumor Immunotherapy.

PubMed

Michen, Susanne; Temme, Achim

2016-01-01

Natural killer (NK) cells are lymphoid cells of the innate immune system; they stand at the first defense line against viruses and transformed cells. NK cells use an array of germline-encoded activating and inhibitory receptors that sense virus-infected cells or malignant cells displaying altered surface expression of activating and inhibitory NK cell ligands. They exert potent cytotoxic responses to cellular targets and thus are candidate effector cells for immunotherapy of cancer. In particular, the genetic engineering of NK cells with chimeric antigen receptors (CARs) against surface-expressed tumor-associated antigens (TAAs) seems promising. In the allogeneic context, gene-modified NK cells compared to T cells may be superior because they are short-lived effector cells and do not cause graft-versus-host disease. Furthermore, their anti-tumoral activity can be augmented by combinatorial use with therapeutic antibodies, chemotherapeutics, and radiation. Today, efforts are being undertaken for large-scale NK-cell expansion and their genetic engineering for adoptive cell transfer. With the recent advances in understanding the complex biological interactions that regulate NK cells, it is expected that the genetic engineering of NK cells and a combinatorial blockade of immune evasion mechanisms are required to exploit the full potential of NK-cell-based immunotherapies.

Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

PubMed

Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

2018-06-01

A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells.

PubMed

Hatziapostolou, Maria; Polytarchou, Christos; Katsoris, Panagiotis; Courty, Jose; Papadimitriou, Evangelia

2006-10-27

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.

N-acetylcysteine negatively regulates Notch3 and its malignant signaling

PubMed Central

Zhu, Juan-Juan; Liu, Xue-Xia; You, Hui; Gong, Mei-Ying; Zou, Ming; Cheng, Wen-Hsing; Zhu, Jian-Hong

2016-01-01

Notch3 receptor is expressed in a variety of cancers and the excised active intracellular domain (N3ICD) initiates its signaling cascade. N-acetylcysteine (NAC) as an antioxidant has been implicated in cancer prevention and therapy. In this study, we demonstrated a negative regulation of Notch3 by NAC in cancer cells. HeLa cells treated with NAC exhibited a time- and concentration-dependent decrease in Notch3 levels and its downstream effectors Hes1 and HRT1 in a manner independent of f-secretase or glutathione. In contrast, NAC did not affect protein levels of Notch1, the full length Notch3 precursor, or ectopically expressed N3ICD. Although SOD, catalase and NAC suppressed reactive oxygen species in HeLa cells, the first two antioxidants did not impact on Notch3 levels. While the mRNA expression of Notch3 was not altered by NAC, functional inhibition of lysosome, but not proteasome, blocked the NAC-dependent reduction of Notch3 levels. Furthermore, results from Notch3 silencing and N3ICD overexpression demonstrated that NAC prevented malignant phenotypes through down-regulation of Notch3 protein in multiple cancer cells. In summary, NAC reduces Notch3 levels through lysosome-dependent protein degradation, thereby negatively regulates Notch3 malignant signaling in cancer cells. These results implicate a novel NAC treatment in sensitizing Notch3-expressing tumors. PMID:27102435

N-acetylcysteine negatively regulates Notch3 and its malignant signaling.

PubMed

Zhang, Xiong; Wang, Ya-Nan; Zhu, Juan-Juan; Liu, Xue-Xia; You, Hui; Gong, Mei-Ying; Zou, Ming; Cheng, Wen-Hsing; Zhu, Jian-Hong

2016-05-24

Notch3 receptor is expressed in a variety of cancers and the excised active intracellular domain (N3ICD) initiates its signaling cascade. N-acetylcysteine (NAC) as an antioxidant has been implicated in cancer prevention and therapy. In this study, we demonstrated a negative regulation of Notch3 by NAC in cancer cells. HeLa cells treated with NAC exhibited a time- and concentration-dependent decrease in Notch3 levels and its downstream effectors Hes1 and HRT1 in a manner independent of f-secretase or glutathione. In contrast, NAC did not affect protein levels of Notch1, the full length Notch3 precursor, or ectopically expressed N3ICD. Although SOD, catalase and NAC suppressed reactive oxygen species in HeLa cells, the first two antioxidants did not impact on Notch3 levels. While the mRNA expression of Notch3 was not altered by NAC, functional inhibition of lysosome, but not proteasome, blocked the NAC-dependent reduction of Notch3 levels. Furthermore, results from Notch3 silencing and N3ICD overexpression demonstrated that NAC prevented malignant phenotypes through down-regulation of Notch3 protein in multiple cancer cells. In summary, NAC reduces Notch3 levels through lysosome-dependent protein degradation, thereby negatively regulates Notch3 malignant signaling in cancer cells. These results implicate a novel NAC treatment in sensitizing Notch3-expressing tumors.

Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

PubMed

Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

2016-01-01

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.

Characterization of a full-length infectious cDNA clone and a GFP reporter derivative of the oncolytic picornavirus SVV-001.

PubMed

Poirier, John T; Reddy, P Seshidhar; Idamakanti, Neeraja; Li, Shawn S; Stump, Kristine L; Burroughs, Kevin D; Hallenbeck, Paul L; Rudin, Charles M

2012-12-01

Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.

Tissue plasminogen activator (tPA) as a reporter gene in transient gene expression.

PubMed

Cheng, S M; Lee, S G; Kalyan, N K; McCloud, S; Levner, M; Hung, P P

1987-01-01

Using the gene coding for tissue plasminogen activator (tPA) as a reporter gene, a transient gene expression system has been established. Vectors containing the full-length cDNA of tPA with its signal sequences were introduced into mammalian recipient cells by a modified gene transfer procedure. Thirty hours after transfection, the secreted tPA was found in serum-free medium and measured by a fibrin-agarose plate assay (FAPA). In this assay, tPA converts plasminogen into plasmin which then degrades high-Mr fibrin to produce cleared zones. The sizes of these zones correspond to quantities of tPA. The combination of transient tPA expression system and the FAPA provides a quick, sensitive, quantitative and non-destructive method to examine the strength of eukaryotic regulatory elements in tissue-culture cells.

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - β1 (TGF-β1) and potential effects on migration and invasion.

PubMed

Magnussen, Synnove Norvoll; Hadler-Olsen, Elin; Costea, Daniela Elena; Berg, Eli; Jacobsen, Cristiane Cavalcanti; Mortensen, Bente; Salo, Tuula; Martinez-Zubiaurre, Inigo; Winberg, Jan-Olof; Uhlin-Hansen, Lars; Svineng, Gunbjorg

2017-05-19

Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. These results show that soluble factors in the tumour microenvironment, such as TGF-β1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

Gene network analysis identifies rumen epithelial cell proliferation, differentiation and metabolic pathways perturbed by diet and correlated with methane production

PubMed Central

Xiang, Ruidong; McNally, Jody; Rowe, Suzanne; Jonker, Arjan; Pinares-Patino, Cesar S.; Oddy, V. Hutton; Vercoe, Phil E.; McEwan, John C.; Dalrymple, Brian P.

2016-01-01

Ruminants obtain nutrients from microbial fermentation of plant material, primarily in their rumen, a multilayered forestomach. How the different layers of the rumen wall respond to diet and influence microbial fermentation, and how these process are regulated, is not well understood. Gene expression correlation networks were constructed from full thickness rumen wall transcriptomes of 24 sheep fed two different amounts and qualities of a forage and measured for methane production. The network contained two major negatively correlated gene sub-networks predominantly representing the epithelial and muscle layers of the rumen wall. Within the epithelium sub-network gene clusters representing lipid/oxo-acid metabolism, general metabolism and proliferating and differentiating cells were identified. The expression of cell cycle and metabolic genes was positively correlated with dry matter intake, ruminal short chain fatty acid concentrations and methane production. A weak correlation between lipid/oxo-acid metabolism genes and methane yield was observed. Feed consumption level explained the majority of gene expression variation, particularly for the cell cycle genes. Many known stratified epithelium transcription factors had significantly enriched targets in the epithelial gene clusters. The expression patterns of the transcription factors and their targets in proliferating and differentiating skin is mirrored in the rumen, suggesting conservation of regulatory systems. PMID:27966600

Expression and characterization of codon-optimized Crimean-Congo hemorrhagic fever virus Gn glycoprotein in insect cells.

PubMed

Rahpeyma, Mehdi; Samarbaf-Zadeh, Alireza; Makvandi, Manoochehr; Ghadiri, Ata A; Dowall, Stuart D; Fotouhi, Fatemeh

2017-07-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a major cause of tick-borne viral hemorrhagic disease in the world. Despite of its importance as a deadly pathogen, there is currently no licensed vaccine against CCHF disease. The attachment glycoprotein of CCHFV (Gn) is a potentially important target for protective antiviral immune responses. To characterize the expression of recombinant CCHFV Gn in an insect-cell-based system, we developed a gene expression system expressing the full-length coding sequence under a polyhedron promoter in Sf9 cells using recombinant baculovirus. Recombinant Gn was purified by affinity chromatography, and the immunoreactivity of the protein was evaluated using sera from patients with confirmed CCHF infection. Codon-optimized Gn was successfully expressed, and the product had the expected molecular weight for CCHFV Gn glycoprotein of 37 kDa. In time course studies, the optimum expression of Gn occurred between 36 and 48 hours postinfection. The immunoreactivity of the recombinant protein in Western blot assay against human sera was positive and was similar to the results obtained with the anti-V5 tag antibody. Additionally, mice were subjected to subcutaneous injection with recombinant Gn, and the cellular and humoral immune response was monitored. The results showed that recombinant Gn protein was highly immunogenic and could elicit high titers of antigen-specific antibodies. Induction of the inflammatory cytokine interferon-gamma and the regulatory cytokine IL-10 was also detected. In conclusion, a recombinant baculovirus harboring CCHFV Gn was constructed and expressed in Sf9 host cells for the first time, and it was demonstrated that this approach is a suitable expression system for producing immunogenic CCHFV Gn protein without any biosafety concerns.

Cancer-associated variant expression and interaction of CIZ1 with cyclin A1 in differentiating male germ cells.

PubMed

Greaves, Erin A; Copeland, Nikki A; Coverley, Dawn; Ainscough, Justin F X

2012-05-15

CIZ1 is a nuclear-matrix-associated DNA replication factor unique to higher eukaryotes, for which alternatively spliced isoforms have been associated with a range of disorders. In vitro, the CIZ1 N-terminus interacts with cyclin E and cyclin A at distinct sites, enabling functional cooperation with cyclin-A-Cdk2 to promote replication initiation. C-terminal sequences anchor CIZ1 to fixed sites on the nuclear matrix, imposing spatial constraint on cyclin-dependent kinase activity. Here we demonstrate that CIZ1 is predominantly expressed as a predicted full-length product throughout mouse development, consistent with a ubiquitous role in cell and tissue renewal. CIZ1 is expressed in proliferating stem cells of the testis, but is notably downregulated following commitment to differentiation. Significantly, CIZ1 is re-expressed at high levels in non-proliferative spermatocytes before meiotic division. Sequence analysis identifies at least seven alternatively spliced variants, including a dominant cancer-associated form and a set of novel isoforms. Furthermore, we show that in these post-replicative cells, CIZ1 interacts with germ-cell-specific cyclin A1, which has been implicated in the repair of DNA double-strand breaks. Consistent with this role, antibody depletion of CIZ1 reduces the capacity for testis extract to repair digested plasmid DNA in vitro. Together, the data imply post-replicative roles for CIZ1 in germ cell differentiation that might include meiotic recombination - a process intrinsic to genome stability and diversification.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolloskis, Michael P.; Carvalho, Fabiana P.; Loo, George, E-mail: g_loo@uncg.edu

Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO),more » PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases. - Highlights: • PEITC increased HO-1 expression in HCT116 cells. • PEITC-induced HO-1 upregulation was impaired in iron-depleted HCT116 cells. • Impairment of PEITC-induced HO-1 upregulation was reversible with iron restoration. • PEITC increased nuclear expression of Nrf2 but not in iron-depleted cells. • NADPH oxidase inhibitors inhibited PEITC-induced HO-1 upregulation.« less

Double-filter identification of vascular-expressed genes using Arabidopsis plants with vascular hypertrophy and hypotrophy.

PubMed

Ckurshumova, Wenzislava; Scarpella, Enrico; Goldstein, Rochelle S; Berleth, Thomas

2011-08-01

Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells

PubMed Central

Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z.; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung’u, Thumbi; Walker, Bruce D.; Rosenberg, Eric S.; Yu, Xu G.

2017-01-01

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells. PMID:28628034

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

PubMed

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

A novel RET rearrangement (ACBD5/RET) by pericentric inversion, inv(10)(p12.1;q11.2), in papillary thyroid cancer from an atomic bomb survivor exposed to high-dose radiation.

PubMed

Hamatani, Kiyohiro; Eguchi, Hidetaka; Koyama, Kazuaki; Mukai, Mayumi; Nakachi, Kei; Kusunoki, Yoichiro

2014-11-01

During analysis of RET/PTC rearrangements in papillary thyroid cancer (PTC) among atomic bomb survivors, a cDNA fragment of a novel type of RET rearrangement was identified in a PTC patient exposed to a high radiation dose using the improved 5' RACE method. This gene resulted from the fusion of the 3' portion of RET containing tyrosine kinase domain to the 5' portion of the acyl-coenzyme A binding domain containing 5 (ACBD5) gene, by pericentric inversion inv(10)(p12.1;q11.2); expression of the fusion gene was confirmed by RT-PCR. ACBD5 gene is ubiquitously expressed in various human normal tissues including thyroid. Full-length cDNA of the ACBD5-RET gene was constructed and then examined for tumorigenicity. Enhanced phosphorylation of ERK proteins in the MAPK pathway was observed in NIH3T3 cells transfected with expression vector encoding the full-length ACBD5/RET cDNA, while this was not observed in the cells transfected with empty expression vector. Stable NIH3T3 transfectants with ACBD5-RET cDNA induced tumor formation after their injection into nude mice. These findings suggest that the ACBD5-RET rearrangement is causatively involved in the development of PTC.

Gelsolin functions as a metastasis suppressor in B16-BL6 mouse melanoma cells and requirement of the carboxyl-terminus for its effect.

PubMed

Fujita, H; Okada, F; Hamada , J; Hosokawa, M; Moriuchi, T; Koya, R C; Kuzumaki, N

2001-09-01

Gelsolin, an actin-binding protein, is implicated as a critical regulator in cell motility. In addition, we have reported that cellular levels of gelsolin are decreased in various tumor cells, and overexpression of gelsolin by gene transfer suppresses tumorigenicity. We sought to assess the effects of gelsolin overexpression on metastasis and to determine the importance of a carboxyl-terminus that confers Ca(2+) dependency on gelsolin for effects of its overexpression. Expression vectors with cDNA encoding either full-length wild-type or His321 mutant form, isolated from a flat revertant of Ras-transformed cells and a carboxyl-terminal truncate, C-del of gelsolin, were transfected into a highly metastatic murine melanoma cell line, B16-BL6. Expression of introduced cDNA in transfectants was confirmed using Western blotting, 2-dimensional gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR). We characterized phenotypes of transfectants, such as growth rate, colony formation in soft agar, cell motility and metastasis formation in vivo. Transfectants expressing the wild-type, His321 mutant and C-del gelsolin exhibited reduced growth ability in soft agar. Although expression of integrin beta1 or alpha4 on the cell surface of transfectants was not changed, wild-type and His321 mutant gelsolin, except for C-del gelsolin, exhibited retardation of cell spreading, reduced chemotatic migration to fibronectin and suppressed lung colonization in spontaneous metastasis assay. Gelsolin may function as a metastasis suppressor as well as a tumor suppressor gene. The carboxyl-terminus of gelsolin is important for retardation of cell spreading, reduced chemotasis and metastasis suppression. Copyright 2001 Wiley-Liss, Inc.

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

PubMed

Roy, Ankita; Goodman, Joshua H; Begum, Gulnaz; Donnelly, Bridget F; Pittman, Gabrielle; Weinman, Edward J; Sun, Dandan; Subramanya, Arohan R

2015-02-15

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors

PubMed Central

Giritch, Anatoli; Marillonnet, Sylvestre; Engler, Carola; van Eldik, Gerben; Botterman, Johan; Klimyuk, Victor; Gleba, Yuri

2006-01-01

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events. PMID:16973752

The expression of MCM-2 in invasive breast carcinoma: a stereologic approach.

PubMed

Cobanoglu, Umit; Mungan, Sevdegul; Gundogdu, Cemal; Ersoz, Safak; Ozoran, Yavuz; Aydin, Fazil

2010-01-01

The aim of this study is to examine the expression of MCM-2 and conventional proliferation marker Ki-67 in breast carcinoma by stereologic technique and to compare it with various clinicopathologic parameters. The expression of MCM-2 and Ki-67 on paraffin-embedded tumor tissue sections of patients with invasive breast carcinoma was analyzed immunohistochemically. Stereologic method was used for evaluation of the percentage of positively stained tumor cells. Significant positive correlation was found between the expression of MCM-2 and that of Ki-67 (r = 0.74, p < 0.001). MCM-2 and Ki-67 expression was significantly associated with histologic grade (p < 0.05), and negative correlation was observed between MCM-2 or Ki-67 expression and estrogen status (p < 0.05). No significant association was observed between MCM-2 or Ki-67 expression and patient age, tumor size, lymph node status, clinical stage and menopausal status. Our results suggest that MCM-2 expression is significantly associated with histologic grade of breast carcinoma and with cell proliferation capacity (Ki-67 labelling index). Additional studies are required using the stereologic method to compare and understand the utility of Ki-67 and MCM-2 expression in invasive breast carcinoma (Tab. 1, Fig. 4, Ref. 34). Full Text (Free, PDF) www.bmj.sk.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodyer, P.R.; Torban, E.; Dehbi, M.

1994-09-01

The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-lengthmore » WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.« less

Meta-analysis of Clear Cell Renal Cell Carcinoma Gene Expression Defines a Variant Subgroup and Identifies Gender Influences on Tumor Biology

PubMed Central

Brannon, A. Rose; Haake, Scott M.; Hacker, Kathryn E.; Pruthi, Raj S.; Wallen, Eric M.; Nielsen, Matthew E.; Rathmell, W. Kimryn

2011-01-01

Background Clear cell renal cell carcinoma (ccRCC) displays molecular and histologic heterogeneity. Previously described subsets of this disease, ccA and ccB, were defined based on multigene expression profiles, but it is unclear whether these subgroupings reflect the full spectrum of disease or how these molecular subtypes relate to histologic descriptions or gender. Objective Determine whether additional subtypes of ccRCC exist and whether these subtypes are related to von Hippel-Lindau (VHL) inactivation, hypoxia-inducible factor (HIF) 1 and 2 expression, tumor histology, or gender. Design, setting, and participants Six large, publicly available ccRCC gene expression databases were identified that cumulatively provided data for 480 tumors for meta-analysis via meta-array compilation. Measurements Unsupervised consensus clustering was performed on the meta-arrays. Tumors were examined for the relationship of multigene-defined consensus subtypes and expression signatures of VHL mutation and HIF status, tumor histology, and gender. Results and limitations Two dominant subsets of ccRCC were observed. However, a minor third cluster was revealed that correlated strongly with a wild type (WT) VHL expression profile and indications of variant histologies. When variant histologies were removed, ccA tumors naturally divided by gender. This technique is limited by the potential for persistent batch effect, tumor sampling bias, and restrictions of annotated information. Conclusions The ccA and ccB subsets of ccRCC are robust in meta-analysis among histologically conventional ccRCC tumors. A third group of tumors was identified that may represent a new variant of ccRCC. Within definitively clear cell tumors, gender may delineate tumors in such a way that it could have implications regarding current treatments and future drug development. PMID:22030119

Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function

PubMed Central

Gimond, Clotilde; van der Flier, Arjan; van Delft, Sanne; Brakebusch, Cord; Kuikman, Ingrid; Collard, John G.; Fässler, Reinhard; Sonnenberg, Arnoud

1999-01-01

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and β1 integrins influence each other using two different β1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of β1A or the cytoplasmic splice variant β1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of β1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of β1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-β1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM–cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length β1A. This indicates that the disruption of cell–cell adhesion is not simply the consequence of the stimulated cell migration. Expression of β1 integrins in GE11 cells resulted in a decrease in cadherin and α-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of α-catenin protein levels by β1 integrins is likely to play a role in the morphological transition, since overexpression of α-catenin in GE11 cells before β1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of β1A, β1D, or IL2R-β1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell–cell adhesions when expressed before β1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-β1A cells. Our results indicate that β1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of α-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA. PMID:10601344

Agonist Met antibodies define the signalling threshold required for a full mitogenic and invasive program of Kaposi's Sarcoma cells.

PubMed

Bardelli, Claudio; Sala, Marilena; Cavallazzi, Umberto; Prat, Maria

2005-09-09

We previously showed that the Kaposi Sarcoma line KS-IMM express a functional Met tyrosine kinase receptor, which, upon HGF stimulation, activates motogenic, proliferative, and invasive responses. In this study, we investigated the signalling pathways activated by HGF, as well as by Met monoclonal antibodies (Mabs), acting as full or partial agonists. The full agonist Mab mimics HGF in all biological and biochemical aspects. It elicits the whole spectrum of responses, while the partial agonist Mab induces only wound healing. These differences correlated with a more prolonged and sustained tyrosine phosphorylation of the receptor and MAPK evoked by HGF and by the full agonist Mab, relative to the partial agonist Mab. Since Gab1, JNK and PI 3-kinase are activated with same intensity and kinetics by HGF and by the two agonist antibodies, it is concluded that level and duration of MAPK activation by Met receptor are crucial for the induction of a full HGF-dependent mitogenic and invasive program in KS cells.

Hematopoietic Colony Formation from Human Growth Factor-Dependent TF1 Cells and Human Cord Blood Myeloid Progenitor Cells Depends on SHP2 Phosphatase Function

PubMed Central

Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E.; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun

2013-01-01

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity. PMID:23082805

Hematopoietic colony formation from human growth factor-dependent TF1 cells and human cord blood myeloid progenitor cells depends on SHP2 phosphatase function.

PubMed

Broxmeyer, Hal E; Etienne-Julan, Maryse; Gotoh, Akihiko; Braun, Stephen E; Lu, Li; Cooper, Scott; Feng, Gen-Sheng; Li, Xing Jun; Chan, Rebecca J

2013-03-15

The protein tyrosine phosphatase, SHP2, is widely expressed; however, previous studies demonstrated that hematopoietic cell development more stringently requires Shp2 expression compared to other tissues. Furthermore, somatic gain-of-function SHP2 mutants are commonly found in human myeloid leukemias. Given that pharmacologic inhibitors to SHP2 phosphatase activity are currently in development as putative antileukemic agents, we conducted a series of experiments examining the necessity of SHP2 phosphatase activity for human hematopoiesis. Anti-sense oligonucleotides to human SHP2 coding sequences reduced human cord blood- and human cell line, TF1-derived colony formation. Expression of truncated SHP2 bearing its Src homology 2 (SH2) domains, but lacking the phosphatase domain similarly reduced human cord blood- and TF1-derived colony formation. Mechanistically, expression of truncated SHP2 reduced the interaction between endogenous, full-length SHP2 with the adapter protein, Grb2. To verify the role of SHP2 phosphatase function in human hematopoietic cell development, human cord blood CD34+ cells were transduced with a leukemia-associated phosphatase gain-of-function SHP2 mutant or with a phosphatase dead SHP2 mutant, which indicated that increased phosphatase function enhanced, while decreased SHP2 phosphatase function reduced, human cord blood-derived colonies. Collectively, these findings indicate that SHP2 phosphatase function regulates human hematopoietic cell development and imply that the phosphatase component of SHP2 may serve as a pharmacologic target in human leukemias bearing increased SHP2 phosphatase activity.

Barx2 is Expressed in Satellite Cells and is Required for Normal Muscle Growth and Regeneration

PubMed Central

Meech, Robyn; Gonzalez, Katie N.; Barro, Marietta; Gromova, Anastasia; Zhuang, Lizhe; Hulin, Julie-Ann; Makarenkova, Helen P.

2015-01-01

Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2−/− mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2+/− mice. Barx2−/− myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation. PMID:22076929

Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

PubMed Central

Wissing, Silke; Muñoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

2012-01-01

Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

Cell culture compositions

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

2014-03-18

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor.

PubMed

Monaco-Shawver, Linda; Schwartz, Lynnae; Tuluc, Florin; Guo, Chang-Jiang; Lai, Jian Ping; Gunnam, Satya M; Kilpatrick, Laurie E; Banerjee, Pinaki P; Douglas, Steven D; Orange, Jordan S

2011-01-01

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10⁻⁶ M SP and ex vivo NK cells with 10⁻⁵ M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Developmental Considerations of Sperm Protein 17 Gene Expression in Rheumatoid Arthritis Synoviocytes

PubMed Central

Takeoka, Yuichi; Kenny, Thomas P.; Yago, Hisashi; Naiki, Mitsuru; Gershwin, M. Eric; Robbins, Dick L.

2002-01-01

Rheumatoid arthritis (RA) is an autoimmune disease characterized by proliferative synovial tissue. We used mRNA differential display and library subtraction to compare mRNA expression in RA and osteoarthritis (OA) synoviocytes. We initially compared the mRNA expression patterns in 1 female RA and 1 OA synovia and found a differentially expressed 350 bp transcript in the RA synoviocytes which was, by sequence analysis, 100% homologous to sperm protein 17 (Sp17). Moreover, the Sp17 transcript was found differentially expressed in a RA synovial library that was subtracted with an OA synovial library. Using specific primers for full length Sp17, a 1.1 kb transcript was amplified from the synoviocytes of 7 additional female RA patients, sequenced and found to 100% homologous to Sp17. Thus, we found the unexpected expression of Sp17, a thought to be gamete-specific protein, in the synoviocytes of 8/8 female RA patients in contrast to control OA synoviocytes. Interestingly, Sp17's structural relationship with cell-binding and recognition proteins, suggests that Sp17 may function in cell-cell recognition and signaling in the RA synoviocyte. Further, Sp17 could have a significant regulatory role in RA synoviocyte gene transcription and/or signal transduction. Thus, Sp17 could have an important role in RA synoviocyte proliferation or defective apoptosis. Finally, the presence of Sp17 in synoviocytes has interesting developmental considerations. PMID:12739786

Human Papilloma Virus-Dependent HMGA1 Expression Is a Relevant Step in Cervical Carcinogenesis1

PubMed Central

Mellone, Massimiliano; Rinaldi, Christian; Massimi, Isabella; Petroni, Marialaura; Veschi, Veronica; Talora, Claudio; Truffa, Silvia; Stabile, Helena; Frati, Luigi; Screpanti, Isabella; Gulino, Alberto; Giannini, Giuseppe

2008-01-01

HMGA1 is a member of a small family of architectural transcription factors involved in the coordinate assembly of multiprotein complexes referred to as enhanceosomes. In addition to their role in cell proliferation, differentiation, and development, high-mobility group proteins of the A type (HMGA) family members behave as transforming protoncogenes either in vitro or in animal models. Recent reports indicated that HMGA1 might counteract p53 pathway and provided an interesting hint on the mechanisms determining HMGA's transforming potential. HMGA1 expression is deregulated in a very large array of human tumors, including cervical cancer, but very limited information is available on the molecular mechanisms leading to HMGA1 deregulation in cancer cells. Here, we report that HMGA1 expression is sustained by human papilloma virus (HPV) E6/E7 proteins in cervical cancer, as demonstrated by either E6/E7 overexpression or by repression through RNA interference. Knocking down HMGA1 expression by means of RNA interference, we also showed that it is involved in cell proliferation and contributes to p53 inactivation in this type of neoplasia. Finally, we show that HMGA1 is necessary for the full expression of HPV18 E6 and E7 oncoproteins thus establishing a positive autoregulatory loop between HPV E6/E7 and HMGA1 expression. PMID:18670638

Immunological targeting of tumor cells undergoing an epithelial-mesenchymal transition via a recombinant brachyury-yeast vaccine

PubMed Central

Jales, Alessandra; Huang, Bruce; Fernando, Romaine I.; Hodge, James W.; Ardiani, Andressa; Apelian, David

2013-01-01

The embryonic T-box transcription factor brachyury is aberrantly expressed in a range of human tumors. Previous studies have demonstrated that brachyury is a driver of the epithelial-mesenchymal transition (EMT), a process associated with cancer progression. Brachyury expression in human tumor cells enhances tumor invasiveness in vitro and metastasis in vivo, and induces resistance to various conventional therapeutics including chemotherapy and radiation. These characteristics, and the selective expression of brachyury for a range of human tumor types vs. normal adult tissues, make brachyury an attractive tumor target. Due to its intracellular localization and the “undruggable” character of transcription factors, available options to target brachyury are currently limited. Here we report on the development and characterization of an immunological platform for the efficient targeting of brachyury-positive tumors consisting of a heat-killed, recombinant Saccharomyces cerevisiae (yeast)–brachyury vector-based vaccine (designated as GI-6301) that expresses the full-length human brachyury protein. We demonstrate that human dendritic cells treated with recombinant yeast-brachyury can activate and expand brachyury-specific CD4+ and CD8+ T cells in vitro that, in turn, can effectively lyse human tumor cells expressing the brachyury protein. Vaccination of mice with recombinant yeast-brachyury is also shown here to elicit brachyury-specific CD4+ and CD8+ T-cell responses, and to induce anti-tumor immunity in the absence of toxicity. Based on these results, a Phase I clinical trial of GI-6301 is currently ongoing in patients with advanced tumors; to our knowledge, this is the first vaccine platform aimed at targeting a driver of tumor EMT that has successfully reached the clinical stage. PMID:24125763

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

PubMed

Lough, Denver M; Wetter, Nathan; Madsen, Christopher; Reichensperger, Joel; Cosenza, Nicole; Cox, Lisa; Harrison, Carrie; Neumeister, Michael W

2016-02-01

Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6 epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6 epithelial stem cell-seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. Isolated LGR6 epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. Wound beds containing LGR6 stem cell-seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6 stem cell-seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6 stem cell-seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. LGR6 stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6 stem cell-containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell-based construct that is capable of the repair of full-thickness wounds and hair regeneration. Therapeutic, V.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

PubMed Central

Kim, Seong K.; Kim, Seongman; Dai, Gan; Zhang, Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

2012-01-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1,487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. PMID:21794889

Functional and evolutionary insights from the Ciona notochord transcriptome.

PubMed

Reeves, Wendy M; Wu, Yuye; Harder, Matthew J; Veeman, Michael T

2017-09-15

The notochord of the ascidian Ciona consists of only 40 cells, and is a longstanding model for studying organogenesis in a small, simple embryo. Here, we perform RNAseq on flow-sorted notochord cells from multiple stages to define a comprehensive Ciona notochord transcriptome. We identify 1364 genes with enriched expression and extensively validate the results by in situ hybridization. These genes are highly enriched for Gene Ontology terms related to the extracellular matrix, cell adhesion and cytoskeleton. Orthologs of 112 of the Ciona notochord genes have known notochord expression in vertebrates, more than twice as many as predicted by chance alone. This set of putative effector genes with notochord expression conserved from tunicates to vertebrates will be invaluable for testing hypotheses about notochord evolution. The full set of Ciona notochord genes provides a foundation for systems-level studies of notochord gene regulation and morphogenesis. We find only modest overlap between this set of notochord-enriched transcripts and the genes upregulated by ectopic expression of the key notochord transcription factor Brachyury, indicating that Brachyury is not a notochord master regulator gene as strictly defined. © 2017. Published by The Company of Biologists Ltd.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

Irisin inhibition of growth hormone secretion in cultured tilapia pituitary cells.

PubMed

Lian, Anji; Li, Xin; Jiang, Quan

2017-01-05

Irisin, the product of fibronectin type III domain-containing protein 5 (FNDC5) gene, is well-documented to be a regulator of energy metabolism. At present, not much is known about its biological function in non-mammalian species. In this study, a full-length tilapia FDNC5 was cloned and its tissue expression pattern has been confirmed. Based on the sequence obtained, we produced and purified recombinant irisin which could induce uncoupling protein 1 (UCP1) gene expression in tilapia hepatocytes. Further, the rabbit polyclonal irisin antiserum was produced and its specificity was confirmed by antiserum preabsorption. In tilapia pituitary cells, irisin inhibited growth hormone (GH) gene expression and secretion and triggered rapid phosphorylation of Akt, Erk1/2, and p38 MAPK. Furthermore, irisin-inhibited GH mRNA expression could be prevented by inhibiting PI3K/Akt, MEK1/2, and p38 MAPK, respectively. Apparently, fish irisin can act directly at the pituitary level to inhibit GH transcript expression via multiple signaling pathways. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

MED1 mediates androgen receptor splice variant induced gene expression in the absence of ligand

PubMed Central

Liu, Gang; Sprenger, Cynthia; Wu, Pin-Jou; Sun, Shihua; Uo, Takuma; Haugk, Kathleen; Epilepsia, Kathryn Soriano; Plymate, Stephen

2015-01-01

The appearance of constitutively active androgen receptor splice variants (AR-Vs) has been proposed as one of the causes of castration-resistant prostate cancer (CRPC). However, the underlying mechanism of AR-Vs in CRPC transcriptional regulation has not been defined. A distinct transcriptome enriched with cell cycle genes, e.g. UBE2C, has been associated with AR-Vs, which indicates the possibility of an altered transcriptional mechanism when compared to full-length wild-type AR (ARfl). Importantly, a recent study reported the critical role of p-MED1 in enhancing UBE2C expression through a locus looping pattern, which only occurs in CRPC but not in androgen-dependent prostate cancer (ADPC). To investigate the potential correlation between AR-V and MED1, in the present study we performed protein co-immunoprecipitation, chromatin immunoprecipitation, and cell proliferation assays and found that MED1 is necessary for ARv567es induced UBE2C up-regulation and subsequent prostate cancer cell growth. Furthermore, p-MED1 is bound to ARv567es independent of full-length AR; p-MED1 has higher recruitment to UBE2C promoter and enhancer regions in the presence of ARv567es. Our data indicate that p-MED1 serves as a key mediator in ARv567es induced gene expression and suggests a mechanism by which AR-Vs promote the development and progression of CRPC. PMID:25481872

Human trophoblast cell during first trimester after IVF-ET differs from natural conceived pregnancy in development and function.

PubMed

Yang, Rui; Liu, Ying-Ying; Zhao, Liang; Wang, Ying; Li, Rong; Liu, Ping; Ma, Cai-Hong; Chen, Xin-Na; Qiao, Jie

2017-03-01

To explore the differences of the trophoblast cell function in first trimester between natural pregnancy and pregnancy after IVF-ET therapy. 102 cases with twin to singleton fetal reduction after IVF-ET treatment from July 2010 to August 2013 in Peking University Third Hospital were involved in analysis, and eight specimens were obtained from this group. 10 natural-pregnancy cases undergoing artificial abortion with unwanted pregnancy were recruited as control. Semi-quantitative immunohistochemical method was used to detect the expression of EGFR, Bcl-2, tubulin-α, metallothionein and AFP in villi in both groups. Of the 102 cases, 14 cases (13.73%) were aborted. Preterm birth occurred in seven cases (7.86%). Low birth weight occurred in three patients (3.37%), and extremely low birth weight occurred in four cases (4.49%). The expression of EGFR, tubulin-α, Bcl-2, and metallothionein in the IVF-ET group was significantly lower than that in the control group (P<0.05). However, AFP expression was significantly higher in IVF-ET group than in control group (P<0.05). In IVF-ET group, the miscarriage case had weaker EGFR, tubulin-α, and metallothionein expression than full-term pregnancy; the early preterm labor case had weaker Bcl-2, tubulin-α, and metallothionein expression; and velamentous cord insertion case had weaker tubulin-α expression. The trophoblast cell function of IVF-ET group in first trimester is different from control group in proliferation, invasion, apoptosis and vascular development, and optimal pregnancy outcome depends on the self-healing balance of trophoblast cells.

Expression and phosphorylation of the AS160_v2 splice variant supports GLUT4 activation and the Warburg effect in multiple myeloma

PubMed Central

2013-01-01

Background Multiple myeloma (MM) is a fatal plasma cell malignancy exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e., the Warburg effect). We have previously demonstrated that myeloma cells exhibit constitutive plasma membrane (PM) localization of GLUT4, consistent with the dependence of MM cells on this transporter for maintenance of glucose consumption rates, proliferative capacity, and viability. The purpose of this study was to investigate the molecular basis of constitutive GLUT4 plasma membrane localization in MM cells. Findings We have elucidated a novel mechanism through which myeloma cells achieve constitutive GLUT4 activation involving elevated expression of the Rab-GTPase activating protein AS160_v2 splice variant to promote the Warburg effect. AS160_v2-positive MM cell lines display constitutive Thr642 phosphorylation, known to be required for inactivation of AS160 Rab-GAP activity. Importantly, we show that enforced expression of AS160_v2 is required for GLUT4 PM translocation and activation in these select MM lines. Furthermore, we demonstrate that ectopic expression of a full-length, phospho-deficient AS160 mutant is sufficient to impair constitutive GLUT4 cell surface residence, which is characteristic of MM cells. Conclusions This is the first study to tie AS160 de-regulation to increased glucose consumption rates and the Warburg effect in cancer. Future studies investigating connections between the insulin/IGF-1/AS160_v2/GLUT4 axis and FDG-PET positivity in myeloma patients are warranted and could provide rationale for therapeutically targeting this pathway in MM patients with advanced disease. PMID:24280290

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gough, Mallory, E-mail: m.gough1@lancaster.ac.uk; Blanthorn-Hazell, Sophee, E-mail: s.blanthorn-hazell@lancaster.ac.uk; Delury, Craig, E-mail: c.delury@lancaster.ac.uk

Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enablemore » cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.« less

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Elucidating the Potential of Plant Rhabdoviruses as Vector Expressions Systems

USDA-ARS?s Scientific Manuscript database

Maize fine streak virus (MFSV) is a member of the genus Nucleorhabdovirus that is transmitted by the leafhopper Graminella nigrifons. The virus replicates in both its maize host and its insect vector. To determine whether Drosophila S2 cells support the production of full-length MFSV proteins, we ...

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

PubMed

Zarei, Najmeh; Vaziri, Behrouz; Shokrgozar, Mohammad Ali; Mahdian, Reza; Fazel, Ramin; Khalaj, Vahid

2014-12-01

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death.

PubMed

Niu, Fangfang; Wang, Chen; Yan, Jingli; Guo, Xiaohua; Wu, Feifei; Yang, Bo; Deyholos, Michael K; Jiang, Yuan-Qing

2016-09-01

NAC transcription factors (TFs) are plant-specific and play important roles in development, responses to biotic and abiotic cues and hormone signaling. So far, only a few NAC genes have been reported to regulate cell death. In this study, we identified and characterized a NAC55 gene isolated from oilseed rape (Brassica napus L.). BnaNAC55 responds to multiple stresses, including cold, heat, abscisic acid (ABA), jasmonic acid (JA) and a necrotrophic fungal pathogen Sclerotinia sclerotiorum. BnaNAC55 has transactivation activity and is located in the nucleus. BnaNAC55 is able to form homodimers in planta. Unlike ANAC055, full-length BnaNAC55, but not either the N-terminal NAC domain or C-terminal regulatory domain, induces ROS accumulation and hypersensitive response (HR)-like cell death when expressed both in oilseed rape protoplasts and Nicotiana benthamiana. Furthermore, BnaNAC55 expression causes obvious nuclear DNA fragmentation. Moreover, quantitative reverse transcription PCR (qRT-PCR) analysis identified that the expression levels of multiple genes regulating ROS production and scavenging, defense response as well as senescence are significantly induced. Using a dual luciferase reporter assay, we further confirm that BnaNAC55 could activate the expression of a few ROS and defense-related gene expression. Taken together, our work has identified a novel NAC TF from oilseed rape that modulates ROS accumulation and cell death.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

PubMed Central

Kamal, Ram P.; Kumar, Amrita; Davis, Charles T.; Tzeng, Wen-Pin; Nguyen, Tung; Donis, Ruben O.; Katz, Jacqueline M.

2015-01-01

ABSTRACT Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses. PMID:25787281

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

PubMed

Kim, Dong-Hyun; Jarvis, Roger M; Allwood, J William; Batman, Gavin; Moore, Rowan E; Marsden-Edwards, Emma; Hampson, Lynne; Hampson, Ian N; Goodacre, Royston

2010-12-01

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

PPARgamma is not a critical mediator of primary monocyte differentiation or foam cell formation.

PubMed

Patel, Lisa; Charlton, Steven J; Marshall, Ian C; Moore, Gary B T; Coxon, Phil; Moores, Kitty; Clapham, John C; Newman, Suzanna J; Smith, Stephen A; Macphee, Colin H

2002-01-18

In the present report we clarify the role of PPARgamma in differentiation and function of human-derived monocyte/macrophages in vitro. Rosiglitazone, a selective PPARgamma activator, had no effect on the kinetics of appearance of monocyte/macrophage differentiation markers or on cell size or granularity. Depletion of PPARgamma by more than 90% using antisense oligonucleotides did not influence accumulation of oxidized LDL or prevent the upregulation of CD36 that normally accompanies oxLDL treatment. In contrast, PPARgamma depletion reduced the expression of ABCA1 and LXRalpha mRNAs. Metalloproteinase-9 expression, a marker of atherosclerotic plaque vulnerability, was suppressed by rosiglitazone. We conclude that activation of PPARgamma does not affect monocyte/macrophage differentiation. In addition, PPARgamma is not absolutely required for oxLDL-driven lipid accumulation, but is required for full expression of ABCA1 and LXRalpha. Our data support a role for rosiglitazone as a potential directly acting antiatherosclerotic agent.

NANOG priming before full reprogramming may generate germ cell tumours.

PubMed

Grad, I; Hibaoui, Y; Jaconi, M; Chicha, L; Bergström-Tengzelius, R; Sailani, M R; Pelte, M F; Dahoun, S; Mitsiadis, T A; Töhönen, V; Bouillaguet, S; Antonarakis, S E; Kere, J; Zucchelli, M; Hovatta, O; Feki, A

2011-11-09

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

PubMed Central

Schemionek, Mirle; Elling, Christian; Steidl, Ulrich; Bäumer, Nicole; Hamilton, Ashley; Spieker, Tilmann; Göthert, Joachim R.; Stehling, Martin; Wagers, Amy; Huettner, Claudia S.; Tenen, Daniel G.; Tickenbrock, Lara; Berdel, Wolfgang E.; Serve, Hubert; Holyoake, Tessa L.; Müller-Tidow, Carsten

2010-01-01

In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity. PMID:20053753

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii.

PubMed

Du, Zhi-Qiang; Lan, Jiang-Feng; Weng, Yu-Ding; Zhao, Xiao-Fan; Wang, Jin-Xing

2013-07-01

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress-induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish. Copyright © 2013 Elsevier Ltd. All rights reserved.

CKIP-1 suppresses odontoblastic differentiation of dental pulp stem cells via BMP2 pathway and can interact with NRP1.

PubMed

Song, Yihua; Wang, Chenfei; Gu, Zhifeng; Cao, Peipei; Huang, Dan; Feng, Guijuan; Lian, Min; Zhang, Ye; Feng, Xingmei; Gao, Zhenran

2018-05-31

Casein kinase 2 interacting protein-1 (CKIP-1) is a recently discovered intracellular regulator of bone formation, muscle cell differentiation and tumor cell proliferation. Our study aims to identify the inhibition of BMP2-Smad1/5 signaling by CKIP-1 in odontoblastic differentiation of human dental pulp stem cells (DPSCs). DPSCs infected CKIP-1 siRNA or transfected CKIP-1 full-length plasmid were cultured in odontoblastic differentiation medium or added noggin (200 ng/mL) for 21 days. We examined the effects of CKIP-1 on odontoblastic differentiation, mineralized nodules formation and interaction by western blot, real-time polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP) staining, alizarin red S staining and immunoprecipitation. Firstly, we have demonstrated that CKIP-1 expression markedly decreased time-dependently along with cell odontoblastic differentiation. Indeed, the silence of CKIP-1 up-regulated odontoblastic differentiation via BMP2-Smad1/5 signaling, while CKIP-1 over-expression had a negative effect on odontoblastic differentiation of DPSCs. Furthermore, CKIP-1 could interact with Neuropilin-1 (NRP1). This work provides data that it advocates a novel perception on odontoblastic differentiation of DPSCs. Therefore, inhibiting the expression of CKIP-1 may be of great significance to the development of dental caries.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

PubMed

Nicolson, Garth L; Nawa, Akihiro; Toh, Yasushi; Taniguchi, Shigeki; Nishimori, Katsuhiko; Moustafa, Amr

2003-01-01

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

C-Type Lectin-Like Receptor 2 Suppresses AKT Signaling and Invasive Activities of Gastric Cancer Cells by Blocking Expression of Phosphoinositide 3-Kinase Subunits.

PubMed

Wang, Lan; Yin, Jie; Wang, Xuefei; Shao, Miaomiao; Duan, Fangfang; Wu, Weicheng; Peng, Peike; Jin, Jing; Tang, Yue; Ruan, Yuanyuan; Sun, Yihong; Gu, Jianxin

2016-05-01

C-type lectin-like receptor 2 (CLEC2) is a transmembrane receptor expressed on platelets and several hematopoietic cells. CLEC2 regulates platelet aggregation and the immune response. We investigated its expression and function in normal and transformed gastric epithelial cells from human tissues. We performed tissue microarray analyses of gastric carcinoma samples collected from 96 patients who underwent surgery at Zhongshan Hospital of Fudan University in Shanghai, China and performed real-time polymerase chain reaction assays from an independent group of 60 patients; matched nontumor gastric mucosa tissues were used as the control. Full-length and mutant forms of CLEC2 were expressed in gastric cancer cell line (MGC80-3), or CLEC2 protein was knocked down using small-hairpin RNAs in gastric cancer cell lines (NCI-N87 and AGS). CLEC2 signaling was stimulated by incubation of cells with recombinant human podoplanin or an antibody agonist of CLEC2; cell migration and invasion were assessed by transwell and wound-healing assays. Immunoblot, immunofluorescence microscopy, and real-time polymerase chain reaction assays were used to measure expression of markers of the epithelial to mesenchymal transition and activation of signaling pathways. Immunoprecipitation experiments were performed with an antibody against spleen tyrosine kinase (SYK). Cells were injected into lateral tail vein of BALB/C nude mice; some mice were also given injections of the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Lung and liver tissues were collected and analyzed for metastases. Levels of CLEC2 were higher in nontumor gastric mucosa (control) than in gastric tumor samples. Levels of CLEC2 protein in gastric tumor tissues correlated with depth of tumor invasion, metastasis to lymph node, tumor TNM stage, and 5-year survival of patients. Activation of CLEC2 in gastric cancer cells reduced their invasive activities in vitro and expression of epithelial to mesenchymal transition markers; these tumor-suppressive effects of CLEC2 required SYK. CLEC2 and SYK interacted physically, and SYK maintained the stability of CLEC2 in cells. AGS cells with CLEC2 knockdown had increased levels of phosphorylated AKT and glycogen synthase kinase-3 beta, increased expression of Snail, reduced levels of E-cadherin, and formed more metastases in mice than AGS cells that expressed CLEC2; these knockdown changes were prevented by the PI3K inhibitor LY294002. Activation of CLEC2 in AGS cells reduced protein and messenger RNA levels of PI3K subunits p85 and p110; this effect was blocked by SYK inhibitor R406. Levels of CLEC2 and SYK proteins and messenger RNAs correlated in gastric tumor samples. CLEC2 suppresses metastasis of gastric cancer cells injected into mice, and prevents activation of AKT and glycogen synthase kinase-3 beta signaling, as well as invasiveness and expression of epithelial to mesenchymal transition markers in gastric cancer cell lines. CLEC2 prevents expression of PI3K subunits, in a SYK-dependent manner. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Potency of umbilical cord blood- and Wharton's jelly-derived mesenchymal stem cells for scarless wound healing.

PubMed

Doi, Hanako; Kitajima, Yuriko; Luo, Lan; Yan, Chan; Tateishi, Seiko; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Mori, Ryoichi; Masuzaki, Hideaki; Shimokawa, Isao; Hirano, Akiyoshi; Li, Tao-Sheng

2016-01-05

Postnatally, scars occur as a consequence of cutaneous wound healing. Scarless wound healing is highly desired for patients who have undergone surgery or trauma, especially to exposed areas. Based on the properties of mesenchymal stem cells (MSCs) for tissue repair and immunomodulation, we investigated the potential of MSCs for scarless wound healing. MSCs were expanded from umbilical cord blood (UCB-MSCs) and Wharton's jelly (WJ-MSCs) from healthy donors who underwent elective full-term pregnancy caesarean sections. UCB-MSCs expressed lower levels of the pre-inflammatory cytokines IL1A and IL1B, but higher levels of the extracellular matrix (ECM)-degradation enzymes MMP1 and PLAU compared with WJ-MSCs, suggesting that UCB-MSCs were more likely to favor scarless wound healing. However, we failed to find significant benefits for stem cell therapy in improving wound healing and reducing collagen deposition following the direct injection of 1.0 × 10(5) UCB-MSCs and WJ-MSCs into 5 mm full-thickness skin defect sites in nude mice. Interestingly, the implantation of UCB-MSCs tended to increase the expression of MMP2 and PLAU, two proteases involved in degradation of the extracellular matrix in the wound tissues. Based on our data, UCB-MSCs are more likely to be a favorable potential stem cell source for scarless wound healing, although a better experimental model is required for confirmation.

TRPC3 Overexpression Promotes the Progression of Inflammation-Induced Preterm Labor and Inhibits T Cell Activation.

PubMed

Jing, Chen; Dongming, Zheng; Hong, Cui; Quan, Na; Sishi, Liu; Caixia, Liu

2018-01-01

To detect the expression of the TRPC3 channel protein in the tissues of women experiencing preterm labor and investigate its interaction with T lymphocytes, providing a theoretical basis for the clinical prevention of threatened preterm labor and the development of drug-targeted therapy. Forty-seven women experiencing preterm labor and 47 women experiencing normal full-term labor were included in this study. All included women underwent delivery via cesarean section; uterine samples were obtained at delivery. The expression of TRPC3 in uterine tissue was detected by immunohistochemistry, real-time quantitative reverse transcription-PCR, and western blot assay. Activation of T lymphocytes in peripheral blood and uterine tissue were detected by flow cytometry. A TRPC3-/- mouse model of inflammation-induced preterm labor was established; expression of TRPC3, Cav3.1, and Cav3.2 were analyzed in mouse uterine tissue. Activation of T lymphocytes in female mouse and human peripheral blood samples was determined using flow cytometry. In women experiencing preterm labor, expression of TRPC3 and the Cav3.1 and Cav3.2 proteins was significantly increased; in addition, the percentage of CD3+, CD4+, and CD8+ T cells in peripheral blood was significantly decreased. TRPC3 knockout significantly delayed the occurrence of preterm labor in mice. The muscle tension of ex vivo uterine strips was lower, Cav3.1 and Cav3.2 protein expression was lower, and the percentage of CD8+ T lymphocytes was significantly increased in wild-type mice subjected to an inflammation-induced preterm labor than in wild-type mice experiencing normal full-term labor. TRPC3 is closely related to the initiation of labor. TRPC3 relies on Cav3.1 and Cav3.2 proteins to inhibit inflammation-induced preterm labor by inhibiting the activation of T cells, in particular CD8+ T lymphocytes. © 2018 The Author(s). Published by S. Karger AG, Basel.

Identification of an anti-lipopolysacchride factor possessing both antiviral and antibacterial activity from the red claw crayfish Cherax quadricarinatus.

PubMed

Lin, Feng-Yu; Gao, Yan; Wang, Hao; Zhang, Qiu-Xia; Zeng, Chang-Lin; Liu, Hai-Peng

2016-10-01

It is well-known that anti-lipopolysacchride factors (ALFs) are involved in the recognition and elimination of invading pathogens. In this study, the full-length ALF cDNA sequence of the red claw crayfish Cherax quadricarinatus (termed CqALF) was cloned from a suppression subtractive hybridization library constructed using red claw crayfish hematopoietic tissue cell (Hpt cell) cultures following challenge with white spot syndrome virus (WSSV). The full-length cDNA sequence of CqALF was 863 bp, and the open reading frame encoded 123 amino acids with a signal peptide in the N-terminus and a conserved LPS-binding domain. Unlike most ALFs, which are highly expressed in haemocytes, high expression levels of CqALF were detected in epithelium, the stomach and eyestalks, while lower expression was detected in Hpt, nerves, the heart, muscle tissue, gonads, haemocytes, intestines, gills and the hepatopancreas. To further explore the biological activities of CqALF, mature recombinant CqALF protein (rCqALF) was expressed and purified using a eukaryotic expression system, and an antimicrobial activity test was carried out. rCqALF clearly exerted antiviral activity, as evidenced by the severe disruption of the envelope of intact WSSV virions following co-incubation of virions with rCqALF. Additionally, pre-incubation of WSSV with rCqALF resulted in both a significant reduction in WSSV replication in red claw crayfish Hpt cell cultures and an increased survival rate among animals. Furthermore, rCqALF was effective against both Gram-negative bacteria and Gram-positive bacteria, particularly Shigella flexneri and Staphylococcus aureus. A membrane integrity assay suggested that rCqALF was unlikely to disrupt bacterial membrane integrity compared to cecropin P1. Taken together, these data suggest that CqALF may play an important role in immune defence in the crustacean C. quadricarinatus. Copyright © 2016 Elsevier Ltd. All rights reserved.

In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

PubMed

Mathivet, Thomas; Mazot, Pierre; Vigny, Marc

2007-12-01

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.

Quantification and study of the L-DOPA decarboxylase expression in gastric adenocarcinoma cells treated with chemotherapeutic substances.

PubMed

Korbakis, Dimitrios; Fragoulis, Emmanuel G; Scorilas, Andreas

2013-03-01

3,4-Dihydroxy-L-phenylalanine decarboxylase (DDC) is an enzyme implicated in the biosynthetic pathways of the neurotransmitters dopamine and probably serotonin. DDC gene expression has been studied in numerous malignancies and the corresponding data have shown remarkable alterations in the mRNA and/or protein levels encoded by the gene. The aim of this study was to examine any modulations in the DDC mRNA levels in gastric cancer cells after their treatment with the chemotherapeutic agents 5-fluorouracil, leucovorin, irinotecan, etoposide, cisplatin, and taxol. The sensitivity of the AGS gastric adenocarcinoma cells to the antineoplastic drugs was evaluated using the MTT assay. Total RNA was extracted and reverse transcribed into cDNA. A highly sensitive quantitative real-time PCR methodology was developed for the quantification of DDC mRNA. GAPDH was used as a housekeeping gene. Relative quantification analysis was carried out using the comparative C T method ((Equation is included in full-text article.)). The treatment of AGS cells with several concentrations of various broadly used anticancer drugs resulted in significant modulations of the DDC mRNA levels compared with those in the untreated cells in a time-specific and drug-specific manner. Generally, DDC expression levels appeared to decrease after three time periods of exposure to the selected chemotherapeutic agents, suggesting a characteristic DDC mRNA expression profile that is possibly related to the mechanism of each drug. Our experimental data show that the DDC gene might serve as a new potential molecular biomarker predicting treatment response in gastric cancer cells.

Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

PubMed

Kanematsu, Daisuke; Shofuda, Tomoko; Yamamoto, Atsuyo; Ban, Chiaki; Ueda, Takafumi; Yamasaki, Mami; Kanemura, Yonehiro

2011-09-01

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine. 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

PubMed

Bayguinov, Peter O; Ma, Yihe; Gao, Yu; Zhao, Xinyu; Jackson, Meyer B

2017-09-20

Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In brain slices from these animals, single-trial hybrid optical voltage sensor recordings revealed voltage changes with submillisecond resolution in multiple neurons simultaneously. This imaging tool will allow for the study of the emergent properties of neural circuits and permit experimental tests of the roles of specific types of neurons in complex circuit activity. Copyright © 2017 the authors 0270-6474/17/379305-15$15.00/0.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth

PubMed Central

Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A.; Andl, Thomas; Zhang, Yuhang

2016-01-01

Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. PMID:27462123

Immortalization of human AE pre-leukemia cells by hTERT allows leukemic transformation

PubMed Central

Wunderlich, Mark; Chou, Fu-Sheng; Mulloy, James C.

2016-01-01

Human CD34+ hematopoietic stem and progenitor cells (HSPC) expressing fusion protein AML1-ETO (AE), generated by the t(8;21)(q22;q22) rearrangement, manifest enhanced self-renewal and dysregulated differentiation without leukemic transformation, representing a pre-leukemia stage. Enabling replicative immortalization via telomerase reactivation is a crucial step in cancer development. However, AE expression alone is not sufficient to maintain high telomerase activity to immortalize human HSPC cells, which may hamper transformation. Here, we investigated the cooperativity of telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, and AE in disease progression. Enforced expression of hTERT immortalized human AE pre-leukemia cells in a telomere-lengthening independent manner, and improved the pre-leukemia stem cell function by enhancing cell proliferation and survival. AE-hTERT cells retained cytokine dependency and multi-lineage differentiation potential similar to parental AE clones. Over the short-term, AE-hTERT cells did not show features of stepwise transformation, with no leukemogenecity evident upon initial injection into immunodeficient mice. Strikingly, after extended culture, we observed full transformation of one AE-hTERT clone, which recapitulated the disease evolution process in patients and emphasizes the importance of acquiring cooperating mutations in t(8;21) AML leukemogenesis. In summary, achieving unlimited proliferative potential via hTERT activation, and thereby allowing for acquisition of additional mutations, is a critical link for transition from pre-leukemia to overt disease in human cells. AE-hTERT cells represent a tractable model to study cooperating genetic lesions important for t(8;21) AML disease progression. PMID:27509060

Taraxacum coreanum protects against glutamate-induced neurotoxicity through heme oxygenase-1 expression in mouse hippocampal HT22 cells.

PubMed

Yoon, Chi-Su; Ko, Wonmin; Lee, Dong-Sung; Kim, Dong-Cheol; Kim, Jongsu; Choi, Moonbum; Beom, Jin Seon; An, Ren-Bo; Oh, Hyuncheol; Kim, Youn-Chul

2017-04-01

Taraxacum coreanum Nakai is a dandelion that is native to Korea, and is widely used as an edible and medicinal herb. The present study revealed the neuroprotective effect of this plant against glutamate-induced oxidative stress in HT22 murine hippocampal neuronal cells. Ethanolic extracts from the aerial (TCAE) and the root parts (TCRE) of T. coreanum were prepared. Both extracts were demonstrated, by high performance liquid chromatography, to contain caffeic acid and ferulic acid as representative constituents. TCAE and TCRE significantly increased cell viability against glutamate-induced oxidative stress in mouse hippocampal HT22 cells. Western blot analysis revealed that treatment of HT22 cells with the extracts induced increased expression of the enzyme heme oxygenase-1 (HO-1), compared with untreated cells, in a concentration-dependent manner. Increased HO-1 enzymatic activity, compared with untreated cells, was also demonstrated following treatment with TCAE and TCRE. In addition, western blot analysis of the nuclear fractions of both TCAE and TCRE-treated HT22 cells revealed increased levels of nuclear factor erythroid 2 like 2 (Nrf2) compared with untreated cells, and decreased Nrf2 levels in the cytoplasmic fraction compared with untreated cells. The present study suggested that the neuroprotective effect of T. coreanum is associated with induction of HO-1 expression and Nrf2 translocation to the nucleus. Therefore, T. coreanum exhibits a promising function in prevention of neurodegeneration. Further studies will be required for the isolation and the full characterization of its active substances.

Cytoskeleton disorder and cell cycle arrest may be associated with the alteration of protein CEP135 by microgravity

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Liu, Zhiyuan

In the past decades, alterations in the morphology, cytoskeleton and cell cycle have been observed in cells in vitro under microgravity conditions. But the underlying mechanisms are not absolutely identified yet. Our previous study on proteomic and microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity has demonstrated a serial of microgravity-sensitive molecules. Centrosomal protein of 135 kDa (CEP135) was found down-regulated, but the mRNA expression level of it was up-regulated in zebrafish embryos after simulated-microgravity. However, the functional study on CEP135 is very limited and it has not been cloned in zebrafish till now. In this study, we try to determine whether the cytoskeleton disorder and cell cycle arrest is associated with the alteration of CEP135 by microgravity. Full-length cDNA of cep135 gene was firstly cloned from mitosis phase of ZF4. The sequence was analyzed and the phylogenetic tree was constructed based on the similarity to other species. Zebrafish embryonic cell line ZF4 were exposed to simulated microgravity for 24 and 48 hours, using a rotary cell culture system (RCCS) designed by NASA. Quantitative analysis by western blot showed that CEP135 expression level was significantly decreased two times after 24 hour simulated microgravity. Cell cycle detection by flow cytometer indicated ZF4 cells were blocked in G1 phase after 24 and 48 hour simulated microgravity. Moreover, double immunostained ZF4 cells with anti-tubulin and anti-CEP135antibodies demonstrated simulated microgravity could lead to cytoskeleton disorder and CEP135 abnormality. Further investigations are currently being carried out to determine whether knockdown and over-expression of CEP135 will modulate cytoskeleton and cell cycle. In vitro data in combination within vivo results might, at least in part, explain the dramatic effects of microgravity. Key Words: microgravity; CEP135; Cytoskeleton disorder; G1 arrest; ZF4 cell line

A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

PubMed

Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

2015-05-01

Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

Communication-dependent mineralization of osteoblasts via gap junctions.

PubMed

Hashida, Yukihiko; Nakahama, Ken-ichi; Shimizu, Kaori; Akiyama, Masako; Harada, Kiyoshi; Morita, Ikuo

2014-04-01

Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2. Copyright © 2013 Elsevier Inc. All rights reserved.

Rapid Development of Salivary Gland Carcinomas upon Conditional Expression of K-ras Driven by the Cytokeratin 5 Promoter

PubMed Central

Raimondi, Ana R.; Vitale-Cross, Lynn; Amornphimoltham, Panomwat; Gutkind, J. Silvio; Molinolo, Alfredo

2006-01-01

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of ΔN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis. PMID:16651631

SHP-2 expression negatively regulates NK cell function1,2

PubMed Central

Purdy, Amanda K.; Campbell, Kerry S.

2009-01-01

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)4 is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human natural killer (NK) cells upon recruitment to killer cell Ig-like receptors (KIR)4. To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-γ production, whereas cells overexpressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center (MTOC)4 polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts MTOC-based cytoskeletal rearrangement and granule release. PMID:19915046

Advanced therapies for the treatment of hemophilia: future perspectives.

PubMed

Liras, Antonio; Segovia, Cristina; Gabán, Aline S

2012-12-13

Monogenic diseases are ideal candidates for treatment by the emerging advanced therapies, which are capable of correcting alterations in protein expression that result from genetic mutation. In hemophilia A and B such alterations affect the activity of coagulation factors VIII and IX, respectively, and are responsible for the development of the disease. Advanced therapies may involve the replacement of a deficient gene by a healthy gene so that it generates a certain functional, structural or transport protein (gene therapy); the incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). For their part, induced pluripotent stem cells have recently been shown to also play a significant role in the fields of cell therapy and tissue engineering. Hemophilia is optimally suited for advanced therapies owing to the fact that, as a monogenic condition, it does not require very high expression levels of a coagulation factor to reach moderate disease status. As a result, significant progress has been possible with respect to these kinds of strategies, especially in the fields of gene therapy (by using viral and non-viral vectors) and cell therapy (by means of several types of target cells). Thus, although still considered a rare disorder, hemophilia is now recognized as a condition amenable to gene therapy, which can be administered in the form of lentiviral and adeno-associated vectors applied to adult stem cells, autologous fibroblasts, platelets and hematopoietic stem cells; by means of non-viral vectors; or through the repair of mutations by chimeric oligonucleotides. In hemophilia, cell therapy approaches have been based mainly on transplantation of healthy cells (adult stem cells or induced pluripotent cell-derived progenitor cells) in order to restore alterations in coagulation factor expression.

Detection of rearrangements and transcriptional up-regulation of ALK in FFPE lung cancer specimens using a novel, sensitive, quantitative reverse transcription polymerase chain reaction assay.

PubMed

Gruber, Kim; Horn, Heike; Kalla, Jörg; Fritz, Peter; Rosenwald, Andreas; Kohlhäufl, Martin; Friedel, Godehard; Schwab, Matthias; Ott, German; Kalla, Claudia

2014-03-01

The approved dual-color fluorescence in situ hybridization (FISH) test for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements in non-small-cell lung cancer (NSCLC) is complex and represents a low-throughput assay difficult to use in daily diagnostic practice. We devised a sensitive and robust routine diagnostic test for the detection of rearrangements and transcriptional up-regulation of ALK. We developed a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay adapted to RNA isolated from routine formalin-fixed, paraffin-embedded material and applied it to 652 NSCLC specimens. The reliability of this technique to detect ALK dysregulation was shown by comparison with FISH and immunohistochemistry. qRT-PCR analysis detected unbalanced ALK expression indicative of a gene rearrangement in 24 (4.6%) and full-length ALK transcript expression in six (1.1%) of 523 interpretable tumors. Among 182 tumors simultaneously analyzed by FISH and qRT-PCR, the latter accurately typed 97% of 19 rearranged and 158 nonrearranged tumors and identified ALK deregulation in two cases with insufficient FISH. Six tumors expressing full-length ALK transcripts did not show rearrangements of the gene. Immunohistochemistry detected ALK protein overexpression in tumors with gene fusions and transcriptional up-regulation, but did not distinguish between the two. One case with full-length ALK expression carried a heterozygous point mutation (S1220Y) within the kinase domain potentially interfering with kinase activity and/or inhibitor binding. Our qRT-PCR assay reliably identifies and distinguishes ALK rearrangements and full-length transcript expression in formalin-fixed, paraffin-embedded material. It is an easy-to-perform, cost-effective, and high-throughput tool for the diagnosis of ALK activation. The expression of full-length ALK transcripts may be relevant for ALK inhibitor therapy in NSCLC.

Osteopontin-a Alters Glucose Homeostasis in Anchorage-Independent Breast Cancer Cells*

PubMed Central

Wang, Bo; Kennedy, Michael A.; Weber, Georg F.

2013-01-01

Invasive breast tumor cells generate three splice variants of the metastasis gene osteopontin, while non-invasive breast cells express only the unspliced form or no osteopontin at all. One role for osteopontin in tumor progression is the support of anchorage-independence. Here we show that the full-length gene product, osteopontin-a, induces a gene expression profile that is associated with tissue remodeling and directed movement/sprouting. This occurs via signals through STAT1 and STAT3 to snglycero-3-phosphocholine. Osteopontin-a upregulates the levels of glucose in breast cancer cells, likely through STAT3 and its transcriptional targets apolipoprotein D and IGFBP5. The splice variants osteopontin-a and osteopontin-c may synergize, with each form activating signal transduction pathways that are distinct from the other. The elevated glucose is used by osteopontin-c dependent signals to generate chemical energy (Shi et al. manuscript submitted). The splice variant-specific metabolic effects of osteopontin add a novel aspect to the pro-metastatic functions of this molecule. PMID:24157812

Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

PubMed Central

Guttman, Mitchell; Garber, Manuel; Levin, Joshua Z.; Donaghey, Julie; Robinson, James; Adiconis, Xian; Fan, Lin; Koziol, Magdalena J.; Gnirke, Andreas; Nusbaum, Chad; Rinn, John L.; Lander, Eric S.; Regev, Aviv

2010-01-01

RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes. PMID:20436462

Probabilistic modeling of bifurcations in single-cell gene expression data using a Bayesian mixture of factor analyzers.

PubMed

Campbell, Kieran R; Yau, Christopher

2017-03-15

Modeling bifurcations in single-cell transcriptomics data has become an increasingly popular field of research. Several methods have been proposed to infer bifurcation structure from such data, but all rely on heuristic non-probabilistic inference. Here we propose the first generative, fully probabilistic model for such inference based on a Bayesian hierarchical mixture of factor analyzers. Our model exhibits competitive performance on large datasets despite implementing full Markov-Chain Monte Carlo sampling, and its unique hierarchical prior structure enables automatic determination of genes driving the bifurcation process. We additionally propose an Empirical-Bayes like extension that deals with the high levels of zero-inflation in single-cell RNA-seq data and quantify when such models are useful. We apply or model to both real and simulated single-cell gene expression data and compare the results to existing pseudotime methods. Finally, we discuss both the merits and weaknesses of such a unified, probabilistic approach in the context practical bioinformatics analyses.

Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV).

PubMed

Wei, Jingguang; Guo, Minglan; Ji, Huasong; Yan, Yang; Ouyang, Zhengliang; Huang, Xiaohong; Hang, Youhua; Qin, Qiwei

2012-10-01

Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

PubMed

Baumgold, J; Spector, I

1987-04-01

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

Phosphatidylethanolamine-binding protein 4 is associated with breast cancer metastasis through Src-mediated Akt tyrosine phosphorylation.

PubMed

Li, H; Huang, F; Fan, L; Jiang, Y; Wang, X; Li, J; Wang, Q; Pan, H; Sun, J; Cao, X; Wang, X

2014-09-11

Metastasis is responsible for more than 90% of the mortality observed among patients with breast cancer. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of the PEBP family and functions as an anti-apoptotic molecule. Here, we found that the metastatic MDA-MB-231 breast cancer cells expressed much higher levels of hPEBP4 than the nonmetastatic MCF-7 breast cancer cells and that the expression levels of hPEBP4 were positively correlated with the metastasis of clinical breast cancer. The hPEBP4 overexpression in the MDA-MB-231 cells significantly promoted cell invasion in vitro and increased the development of lymph node metastasis in vivo. Conversely, the silencing of hPEBP4 suppressed the cell-invasive ability both in vitro and in vivo. Further investigation showed that hPEBP4 promoted the expression or activity of the metastasis-related proteinases MMP (matrix metalloproteinase) 2, MMP9 and MMP13. This hPEBP4-potentiated cell invasion and MMP expression is due to an increase in Akt activation. Knockdown of Akt restored hPEBP4-induced breast tumor metastasis in the hPEBP4-MDA-MB-231 xenograft mouse model. Moreover, we found that hPEBP4 functioned as a scaffolding molecule and enhanced the association of Akt with Src to promote Akt tyrosine phosphorylation, a prerequisite for the full activation of Akt, in a phosphatidylethanolamine-binding domain-dependent manner. Given the present information about human breast cancer, these functional data from cell culture and animal studies suggest that, in human breast cancer hPEBP4 is a novel and clinically relevant metastasis accelerator gene and may be a new diagnostic marker and therapeutic target for breast cancer metastasis.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

PubMed Central

Agrelo, Ruben; Sutz, Miguel Arocena; Setien, Fernando; Aldunate, Fabian; Esteller, Manel; Da Costa, Valeria; Achenbach, Ricardo

2015-01-01

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality. PMID:25830902

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

PubMed

Nika, Lisa; Wallner, Jakob; Palmberger, Dieter; Koczka, Krisztina; Vorauer-Uhl, Karola; Grabherr, Reingard

2017-08-01

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation. Copyright © 2017. Published by Elsevier Inc.

Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits.

PubMed

Gong, Haibiao; Do, Devin; Ramakrishnan, Ramesh

2018-01-01

Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

Does human endometrial LGR5 gene expression suggest the existence of another hormonally regulated epithelial stem cell niche?

PubMed

Tempest, N; Baker, A M; Wright, N A; Hapangama, D K

2018-06-01

Is human endometrial leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) gene expression limited to the postulated epithelial stem cell niche, stratum basalis glands, and is it hormonally regulated? LGR5 expressing cells are not limited to the postulated stem cell niche but LGR5 expression is hormonally regulated. The human endometrium is a highly regenerative tissue; however, endometrial epithelial stem cell markers are yet to be confirmed. LGR5 is a marker of stem cells in various epithelia. The study was conducted at a University Research Institute. Endometrial samples from 50 healthy women undergoing benign gynaecological surgery with no endometrial pathology at the Liverpool Women's hospital were included and analysed in the following six sub-categories; proliferative, secretory phases of menstrual cycle, postmenopausal, those using oral and local progestagens and samples for in vitro explant culture. In this study, we used the gold standard method, in situ hybridisation (ISH) along with qPCR and a systems biology approach to study the location of LGR5 gene expression in full thickness human endometrium and Fallopian tubes. The progesterone regulation of endometrial LGR5 was examined in vivo and in short-term cultured endometrial tissue explants in vitro. LGR5 expression was correlated with epithelial proliferation (Ki67), and expression of previously reported epithelia progenitor markers (SOX9 and SSEA-1) immunohistochemistry (IHC). LGR5 gene expression was significantly higher in the endometrial luminal epithelium than in all other epithelial compartments in the healthy human endometrium, including the endometrial stratum basalis (P < 0.05). The strongest SSEA-1 and SOX9 staining was observed in the stratum basalis glands, but the general trend of SOX9 and SSEA-1 expression followed the same cyclical pattern of expression as LGR5. Stratum functionalis epithelial Ki67-LI and LGR5 expression levels correlated significantly (r = 0.74, P = 0.01), however, they did not correlate in luminal and stratum basalis epithelium (r = 0.5 and 0.13, respectively). Endometrial LGR5 demonstrates a dynamic spatiotemporal expression pattern, suggesting hormonal regulation. Oral and local progestogens significantly reduced endometrial LGR5 mRNA levels compared with women not on hormonal treatment (P < 0.01). Our data were in agreement with in silico analysis of published endometrial microarrays. We did not generate our own large scale data but interrogated publically available large scale data sets. In the absence of reliable antibodies for human LGR5 protein and validated lineage markers for the various epithelial populations that potentially exist within the endometrium, our study does not formally characterise or examine the functional ability of the resident LGR5+ cells as multipotent. These data will facilitate future lineage tracing studies in the human endometrial epithelium; to identify the location of stem cells and further complement the in vitro functional studies, to confirm if the LGR5 expressing epithelial cells indeed represent the epithelial stem cell population. This work was supported by funding from the Wellbeing of Women project grant (RTF510) and Cancer Research UK (A14895). None of the authors have any conflicts of interest to disclose.

Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo: Critical Role of HIF-1α-Neuropilin-1 Axis

PubMed Central

Misra, Roli M.; Bajaj, Manmohan S.; Kale, Vaijayanti P.

2012-01-01

HT1080 - a human fibrosarcoma-derived cell line – forms aggressive angiogenic tumours in immuno-compromised mice. In spite of its extensive use as a model of tumour angiogenesis, the molecular event(s) initiating the angiogenic program in these cells are not known. Since hypoxia stimulates tumour angiogenesis, we examined the hypoxia-induced events evoked in these cells. In contrast to cells grown under normoxic conditions, hypoxia-primed (1% O2) HT1080 cells formed robust tubules on growth factor-reduced matrigel and formed significantly larger tumours in xenograft models in a chetomin-sensitive manner, indicating the role of HIF-1α-mediated transcription in these processes. Immuno-histochemical analyses of tumours formed by GFP-expressing HT1080 cells clearly showed that the tumour cells themselves expressed various angiogenic markers including Neuropilin-1 (NRP-1) and formed functional vessels containing red blood cells, thereby unambiguously demonstrating the vasculogenic mimicry of HT1080 cells in vivo. Experiments performed with the HT1080 cells stably transfected with plasmid constructs expressing shNRP-1 or full-length NRP-1 clearly established that the HIF1α-mediated up-regulation of NRP-1 played a deterministic role in the process. Hypoxia-exposure resulted in an up-regulation of c-Myc and OCT3/4 and a down-regulation of KLF4 mRNAs, suggesting their involvement in the tumour formation and angiogenesis. However, silencing of NRP-1 alone, though not affecting proliferation in culture, was sufficient to abrogate the tumour formation completely; clearly establishing that the hypoxia-mediated HIF-1α-dependent up-regulation of NRP-1 is a critical molecular event involved in the vasculogenic mimicry and tumor formation by HT1080 cells in vivo. PMID:23185562

Identification and characterization of TCRgamma and TCRdelta chains in channel catfish, Ictalurus punctatus

USDA-ARS?s Scientific Manuscript database

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) gamma and delta were identified by mining of expressed sequence tag databases and full length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), (diversity; D), joining ...

The extraction of simple relationships in growth factor-specific multiple-input and multiple-output systems in cell-fate decisions by backward elimination PLS regression.

PubMed

Akimoto, Yuki; Yugi, Katsuyuki; Uda, Shinsuke; Kudo, Takamasa; Komori, Yasunori; Kubota, Hiroyuki; Kuroda, Shinya

2013-01-01

Cells use common signaling molecules for the selective control of downstream gene expression and cell-fate decisions. The relationship between signaling molecules and downstream gene expression and cellular phenotypes is a multiple-input and multiple-output (MIMO) system and is difficult to understand due to its complexity. For example, it has been reported that, in PC12 cells, different types of growth factors activate MAP kinases (MAPKs) including ERK, JNK, and p38, and CREB, for selective protein expression of immediate early genes (IEGs) such as c-FOS, c-JUN, EGR1, JUNB, and FOSB, leading to cell differentiation, proliferation and cell death; however, how multiple-inputs such as MAPKs and CREB regulate multiple-outputs such as expression of the IEGs and cellular phenotypes remains unclear. To address this issue, we employed a statistical method called partial least squares (PLS) regression, which involves a reduction of the dimensionality of the inputs and outputs into latent variables and a linear regression between these latent variables. We measured 1,200 data points for MAPKs and CREB as the inputs and 1,900 data points for IEGs and cellular phenotypes as the outputs, and we constructed the PLS model from these data. The PLS model highlighted the complexity of the MIMO system and growth factor-specific input-output relationships of cell-fate decisions in PC12 cells. Furthermore, to reduce the complexity, we applied a backward elimination method to the PLS regression, in which 60 input variables were reduced to 5 variables, including the phosphorylation of ERK at 10 min, CREB at 5 min and 60 min, AKT at 5 min and JNK at 30 min. The simple PLS model with only 5 input variables demonstrated a predictive ability comparable to that of the full PLS model. The 5 input variables effectively extracted the growth factor-specific simple relationships within the MIMO system in cell-fate decisions in PC12 cells.

Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68▿ †

PubMed Central

Zhu, Jia Yun; Strehle, Martin; Frohn, Anne; Kremmer, Elisabeth; Höfig, Kai P.; Meister, Gunter; Adler, Heiko

2010-01-01

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of γHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of γHV miRNAs. PMID:20668074

Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development.

PubMed

Miller, Nichol L G; Wevrick, Rachel; Mellon, Pamela L

2009-01-15

Prader-Willi syndrome (PWS) is a complex genetic disorder characterized by hyperphagia, obesity and hypogonadotrophic hypogonadism, all highly suggestive of hypothalamic dysfunction. The NDN gene, encoding the MAGE family protein, necdin, maps to the PWS chromosome region and is highly expressed in mature hypothalamic neurons. Adult mice lacking necdin have reduced numbers of gonadotropin-releasing hormone (GnRH) neurons, but the mechanism for this reduction is unknown. Herein, we show that, although necdin is not expressed in an immature, migratory GnRH neuronal cell line (GN11), high levels are present in a mature GnRH neuronal cell line (GT1-7). Furthermore, overexpression of necdin activates GnRH transcription through cis elements bound by the homeodomain repressor Msx that are located in the enhancer and promoter of the GnRH gene, and knock-down of necdin expression reduces GnRH gene expression. In fact, overexpression of Necdin relieves Msx repression of GnRH transcription through these elements and necdin co-immunoprecipitates with Msx from GnRH neuronal cells, indicating that necdin may activate GnRH gene expression by preventing repression of GnRH gene expression by Msx. Finally, necdin is necessary for generation of the full complement of GnRH neurons during mouse development and extension of GnRH axons to the median eminence. Together, these results indicate that lack of necdin during development likely contributes to the hypogonadotrophic hypogonadal phenotype in individuals with PWS.

Necdin, a Prader–Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development

PubMed Central

Miller, Nichol L.G.; Wevrick, Rachel; Mellon, Pamela L.

2009-01-01

Prader–Willi syndrome (PWS) is a complex genetic disorder characterized by hyperphagia, obesity and hypogonadotrophic hypogonadism, all highly suggestive of hypothalamic dysfunction. The NDN gene, encoding the MAGE family protein, necdin, maps to the PWS chromosome region and is highly expressed in mature hypothalamic neurons. Adult mice lacking necdin have reduced numbers of gonadotropin-releasing hormone (GnRH) neurons, but the mechanism for this reduction is unknown. Herein, we show that, although necdin is not expressed in an immature, migratory GnRH neuronal cell line (GN11), high levels are present in a mature GnRH neuronal cell line (GT1-7). Furthermore, overexpression of necdin activates GnRH transcription through cis elements bound by the homeodomain repressor Msx that are located in the enhancer and promoter of the GnRH gene, and knock-down of necdin expression reduces GnRH gene expression. In fact, overexpression of Necdin relieves Msx repression of GnRH transcription through these elements and necdin co-immunoprecipitates with Msx from GnRH neuronal cells, indicating that necdin may activate GnRH gene expression by preventing repression of GnRH gene expression by Msx. Finally, necdin is necessary for generation of the full complement of GnRH neurons during mouse development and extension of GnRH axons to the median eminence. Together, these results indicate that lack of necdin during development likely contributes to the hypogonadotrophic hypogonadal phenotype in individuals with PWS. PMID:18930956

Murine Lupus Susceptibility Locus Sle1a Requires the Expression of two Subloci to Induce Inflammatory T Cells

PubMed Central

Cuda, Carla M.; Zeumer, Leilani; Sobel, Eric S.; Croker, Byron P.; Morel, Laurence

2010-01-01

The NZM2410-derived Sle1a lupus susceptibility locus induces activated autoreactive CD4+ T cells and reduces the number and function of Foxp3+ regulatory T cells. In this study, we first showed that Sle1a contributes to autoimmunity by increasing anti-nuclear antibody production when expressed on either NZB or NZW heterozygous genomes, and by enhancing the chronic graft vs. host disease response indicating an expansion of the autoreactive B cell pool. Screening two non-overlapping recombinants, the Sle1a.1 and Sle1a.2 intervals that cover the entire Sle1a locus, revealed that both Sle1a.1 and Sle1a.2 were necessary for the full Sle1a phenotype. Sle1a.1, and to a lesser extent Sle1a.2, significantly affected CD4+ T cell activation as well as Treg differentiation and function. Sle1a.2 also increased the production of autoreactive B cells. Since the Sle1a.1 and Sle1a.2 intervals contain only one and 15 known genes, respectively, this study considerably reduces the number of candidate genes responsible for the production of autoreactive T cells. These results also demonstrate that the Sle1 locus is an excellent model for the genetic architecture of lupus, in which a major obligate phenotype results from the co-expression of multiple genetic variants with individual weak effects. PMID:20445563

Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

PubMed Central

Sui, Zhiwei; Wen, Bo; Gao, Zhimin; Chen, Quanjiao

2014-01-01

Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses. PMID:25153908

A Role for SMN Exon 7 Splicing in the Selective Vulnerability of Motor Neurons in Spinal Muscular Atrophy

PubMed Central

Ruggiu, Matteo; McGovern, Vicki L.; Lotti, Francesco; Saieva, Luciano; Li, Darrick K.; Kariya, Shingo; Monani, Umrao R.; Burghes, Arthur H. M.

2012-01-01

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1. PMID:22037760

Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity

PubMed Central

Duennwald, Martin L.; Lindquist, Susan

2008-01-01

Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington’s disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin. PMID:19015277

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

PubMed

Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

2016-01-08

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

PubMed Central

Klawitter, Sabine; Fuchs, Nina V.; Upton, Kyle R.; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J.; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J. Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J.; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L.; Faulkner, Geoffrey J.; Schumann, Gerald G.

2016-01-01

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs. PMID:26743714

Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

PubMed Central

Huda, Kazi Md. Kamrul; Banu, Mst. Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

2013-01-01

Background Plasma membrane Ca2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca2+) from the cell, hence regulating Ca2+ level within cells. Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied. Results The 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs. Conclusions The rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology. PMID:23469243

Direct Test for Neuroinflammation with [11C]DAP-713-PET Scanning

DTIC Science & Technology

2016-10-01

support of the PD/PI(s) or senior/key personnel since the last reporting period?  Martin Pomper Ended Title: PSMA -associated PET imaging of CAR T...01/2015 – 10/31/2016 Level of Funding: $95,492 Description of Goals: To develop a PSMA -based molecular-genetic imaging system for tracking T cells...Aim 1: Functional assessment of CAR T cells expressing PSMA (full length and truncated versions) Aim 2: Evaluation of 18F-DCFPyL labelling and

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The PPARγ2 A/B-Domain Plays a Gene-Specific Role in Transactivation and Cofactor Recruitment

PubMed Central

Bugge, Anne; Grøntved, Lars; Aagaard, Mads M.; Borup, Rehannah; Mandrup, Susanne

2009-01-01

We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5–8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARγ2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARγ lacking the A/B-domain (PPARγCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared with cells expressing full-length PPARγ2. Using chromatin immunoprecipitation, we demonstrate that PPARγ binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARγ-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARγ2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes. PMID:19282365

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

PubMed

Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

2012-09-11

Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Elevated numbers of SCART1+ gammadelta T cells in skin inflammation and inflammatory bowel disease.

PubMed

Fink, Dorte Rosenbek; Holm, Dorte; Schlosser, Anders; Nielsen, Ole; Latta, Markus; Lozano, Francisco; Holmskov, Uffe

2010-05-01

The members of the scavenger receptor cysteine-rich (SRCR) superfamily group B have diverse functions, including roles in the immune system. For years it has been known that the WC1 protein is expressed on the surface of bovine gammadelta T cells, and more recent studies indicate that WC1(+) gammadelta T cells respond to stimulation with bacterial antigens by producing interferon-gamma. The SRCR proteins CD5, CD6, Sp alpha, CD163, and DMBT1/gp-340 are also involved in the immune response, since they are pattern recognition receptors capable of binding directly to bacterial and/or fungal components. Here, we investigate a novel murine SRCR protein named SCART1. The ectodomain and the full-length SCART1 were expressed in mammalian cells and used to raise monoclonal antibodies against the ectodomain for immunohistochemical and FACS analysis. Immunohistochemical analysis shows that SCART1 is expressed in a range of lymphoid organs and epithelial-rich tissues by a subset of T cells identified as being gammadelta T cells by FACS analysis. SCART1 was present in 86% of the gammadelta T cells and was not found in CD4(+) or CD8(+) T cells. The numbers of SCART1(+) cells were elevated in two mouse models of human diseases: skin inflammation and inflammatory bowel disease. In the skin inflammation model, an 8.6-fold increase in SCART1(+) cells was observed. Finally, recombinant SCART1 protein was found not to bind to selected bacterial or fungal components or to whole bacteria. Our results show that SCART1 is a novel gammadelta T cell marker and it is therefore likely that SCART1 plays a role in the immune response. (c) 2010 Elsevier Ltd. All rights reserved.

Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

PubMed

Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-01-01

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, Anson, E-mail: piercea2@uthscsa.edu; Barshop Institute for Longevity and Aging Studies, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, 78229; The Department of Veteran's Affairs, South Texas Veterans Health Care System, San Antonio, Texas, 78284

2010-11-05

Research highlights: {yields} Development of mouse overexpressing native human HSF1 in all tissues including CNS. {yields} HSF1 overexpression enhances heat shock response at whole-animal and cellular level. {yields} HSF1 overexpression protects from polyglutamine toxicity and favors aggresomes. {yields} HSF1 overexpression enhances proteostasis at the whole-animal and cellular level. -- Abstract: The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenicmore » mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1{sup +/0}) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1{sup +/0} mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1{sup +/0} cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1{sup +/0} cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.« less

HrpE3 is a type III effector protein required for full virulence of Xanthomonas oryzae pv. oryzicola in rice.

PubMed

Cui, Yiping; Zou, Lifang; Zou, Huasong; Li, Yurong; Zakria, Muhammad; Chen, Gongyou

2013-09-01

Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of bacterial leaf streak, a devastating disease in rice. Xoc uses a type III secretion (T3S) system, which is encoded by the hrp-hrc-hpa (hypersensitive response and pathogenicity, hrp-conserved and hrp-associated) genes, to inject repertoires of T3S effectors (T3Es) into plant cells. Many of the hrp-hrc-hpa genes have roles in pathogenesis, but the role of hrpE3, which shows homology to hpaE in X. campestris pv. vesicatoria (Xcv), is poorly understood. In this study, hrpE3 was shown to be transcribed independent of the hrpD operon, and its expression was dependent on a promoter within hpaB. The expression of hrpE3 was positively regulated by HrpG and HrpX, a finding probably caused by an imperfect plant-inducible promoter (PIP) box (TTCGT-N16 -TTCGA) in the hrpE3 promoter. The secretion of HrpE3 was dependent on T3S, and subcellular localization of HrpE3 was cytoplasmic and nuclear in plant cells. A mutation in hrpE3 reduced the virulence of Xoc by decreasing disease lesion length and bacterial growth in planta. Full virulence was restored to the mutant when Xoc hrpE3, but not Xcv hpaE, was expressed in trans. The differences in transcription, secretion via the T3S system and bacterial virulence in plants were attributed to N-terminal amino acid differences between Xoc HrpE3 and Xcv HpaE. Collectively, the results demonstrate that hrpE3 encodes a T3E protein which is delivered into the plant cell through the T3S system, localizes to the cytoplasm and nucleus, and is required for full virulence in rice. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

PubMed

Rizk, Francine; Laverdure, Sylvain; d'Alençon, Emmanuelle; Bossin, Hervé; Dupressoir, Thierry

2018-01-01

The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV- gfp based molecules which were transfected into non permissive Spodoptera frugiperda ( Sf9 ) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Translational read-through of a nonsense mutation causing Bartter syndrome.

PubMed

Cho, Hee Yeon; Lee, Beom Hee; Cheong, Hae Il

2013-06-01

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics.

PubMed

Buchanan, Paul J; McCloskey, Karen D

2016-10-01

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (Ca V ) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the Ca V channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. Ca V channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several Ca V channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of Ca V channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that Ca V canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of Ca V channels in cancer and whether calcium channel blockers are beneficial in cancer treatment.

Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function*

PubMed Central

Bancroft, Tara; Bouaouina, Mohamed; Roberts, Sophia; Lee, Monica; Calderwood, David A.; Schwartz, Martin; Simons, Michael; Sessa, William C.; Kyriakides, Themis R.

2015-01-01

Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair. PMID:25389299

Dysregulation of chemokine receptor expression and function in leukocytes from ALS patients.

PubMed

Perner, Caroline; Perner, Florian; Stubendorff, Beatrice; Förster, Martin; Witte, Otto W; Heidel, Florian H; Prell, Tino; Grosskreutz, Julian

2018-03-28

Amyotrophic lateral sclerosis (ALS) is rapidly progressive adult-onset motor neuron disease characterized by the neurodegeneration of both upper and lower motor neurons in the cortex and the spinal cord; the majority of patients succumb to respiratory failure. Although the etiology is not yet fully understood, there is compelling evidence that ALS is a multi-systemic disorder, with peripheral inflammation critically contributing to the disease process. However, the full extent and nature of this immunological dysregulation remains to be established, particularly within circulating blood cells. Therefore, the aim of the present study was to identify dysregulated inflammatory molecules in peripheral blood cells of ALS patients and analyze for functional consequences of the observed findings. To this end, we employed flow cytometry-based screening to quantify the surface expression of major chemokine receptors and integrins. A significantly increased expression of CXCR3, CXCR4, CCL2, and CCL5 was observed on T cells in ALS patients compared to healthy controls. Intriguingly, the expression was even more pronounced in patients with a slow progressive phenotype. To further investigate the functional consequences of this altered surface expression, we used a modified Boyden chamber assay to measure chemotaxis in ALS patient-derived lymphocytes. Interestingly, chemoattraction with the CXCR3-Ligand IP10 led to upregulated migratory behavior of ALS lymphocytes compared to healthy controls. Taken together, our data provides evidence for a functional dysregulation of IP10-directed chemotaxis in peripheral blood cells in ALS patients. However, whether the chemokine itself or its receptor CXCR3, or both, could serve as potential therapeutic targets in ALS requires further investigations.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

PubMed

Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

2014-08-01

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Hypodermal decidualized endometrioma with aberrant cytokeratin expression. A lesion mimicking malignancy.

PubMed

Val-Bernal, José Fernando; Val, Daniel; Gómez-Aguado, Fernando; Corcuera, María Teresa; Garijo, María Francisca

2011-07-01

Decidualized endometrioma is a pseudoneoplastic lesion that may appear as a solitary nodule in the hypodermis, simulate a malignant epithelioid tumor, and can represent a diagnostic challenge. A 36-year-old woman delivered a full-term baby by cesarean. At the immediate puerperium, she complained of a subcutaneous nodule measuring 2.5 cm, underneath a previous caesarean scar from the former full-term delivery 3 years earlier. Histologic features included a nodular growth pattern of large monomorphic epithelioid cells showing diffuse positivity for cytokeratin (AE1/AE3, 18), human placental lactogen, and CD10 and focal positivity for inhibin alpha. The main differential diagnoses include trophoblastic neoplasia and deciduoid mesothelioma. Good clinicopathological correlation is essential for the correct diagnosis. Immunohistochemical stains can be misleading. An important clue is the combination of large decidualized cells and lumens lined by flat or low cuboidal cells that are atrophic endometrial glands. This lesion has a benign behavior.

Exogenous DKK-3/REIC inhibits Wnt/β-catenin signaling and cell proliferation in human kidney cancer KPK1.

PubMed

Xu, Jiaqi; Sadahira, Takuya; Kinoshita, Rie; Li, Shun-Ai; Huang, Peng; Wada, Koichiro; Araki, Motoo; Ochiai, Kazuhiko; Noguchi, Hirofumi; Sakaguchi, Masakiyo; Nasu, Yasutomo; Watanabe, Masami

2017-11-01

The third member of the Dickkopf family (DKK-3), also known as reduced expression in immortalized cells (REIC), is a tumor suppressor present in a variety of tumor cells. Regarding the regulation of the Wnt/β-catenin signaling pathway, exogenous DKK-1 and DKK-2 are reported to inhibit Wnt signaling by binding the associated effectors. However, whether exogenous DKK-3 inhibits Wnt signaling remains unclear. A recombinant protein of human full-length DKK-3 was used to investigate the exogenous effects of the protein in vitro in KPK1 human renal cell carcinoma cells. It was demonstrated that the expression of phosphorylated (p-)β-catenin (inactive form as the transcriptional factor) was increased in KPK1 cells treated with the exogenous DKK-3 protein. The levels of non-p-β-catenin (activated form of β-catenin) were consistently decreased. It was revealed that the expression of transcription factor (TCF) 1 and c-Myc, the downstream transcription factors of the Wnt/β-catenin signaling pathway, was inhibited following treatment with DKK-3. A cancer cell viability assay confirmed the anti-proliferative effects of exogenous DKK-3 protein, which was consistent with a suppressed Wnt/β-catenin signaling cascade. In addition, as low-density lipoprotein receptor-related protein 6 (LRP6) is a receptor of DKK-1 and DKK-2 and their interaction on the cell surface inhibits Wnt/β-catenin signaling, it was examined whether the exogenous DKK-3 protein affects LRP6-mediated Wnt/β-catenin signaling. The LRP6 gene was silenced and the effects of DKK-3 on the time course of the upregulation of p-β-catenin expression were subsequently analyzed. Notably, LRP6 depletion elevated the base level of p-β-catenin; however, there was no significant effect on its upregulation course or expression pattern. These findings indicate that exogenous DKK-3 upregulates p-β-catenin and inhibits Wnt/β-catenin signaling in an LRP6-independent manner. Therefore, exogenous DKK-3 protein may inhibit the proliferation of KPK1 cells via inactivating Wnt/β-catenin signaling.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

NF-κB and androgen receptor variant expression correlate with human BPH progression.

PubMed

Austin, David C; Strand, Douglas W; Love, Harold L; Franco, Omar E; Jang, Alex; Grabowska, Magdalena M; Miller, Nicole L; Hameed, Omar; Clark, Peter E; Fowke, Jay H; Matusik, Robert J; Jin, Ren J; Hayward, Simon W

2016-04-01

Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy. © 2015 Wiley Periodicals, Inc.

Corneal endothelial autocrine trophic factor VIP in a mechanism-based strategy to enhance human donor cornea preservation for transplantation.

PubMed

Koh, Shay-Whey Margaret

2012-02-01

Vasoactive intestinal peptide (VIP) and ciliary neurotrophic factor (CNTF) are identified as autocrines of human corneal endothelial (CE) cells working in concert to maintain the differentiated state and promote the survival of the corneal endothelium. From VIP gene knockdown study, endogenous VIP is shown to maintain the level of the differentiation marker, the adhesion molecule N-cadherin, CE cell size, shape, and retention, in situ in the human donor corneoscleral explants. Exogenous VIP protects the corneal endothelium against the killing effect of oxidative stress, in part by upholding ATP levels in CE cells dying of oxidative stress-induced injury, allowing them to die of an apoptotic death instead of an acute necrotic one. The switch from the acute necrosis to the programmed cell death (apoptosis) may have allowed the injured CE cell to be rescued by the VIP-upregulated pathways, including those of Bcl-2 and N-cadherin, and resulted in long-term CE cell survival. The endogenous VIP in CE cells is upregulated by CNTF, which is released by CE cells surviving the oxidative stress. The CNTF receptor (CNTFRα) is expressed in CE cells in human donor corneoscleral explant and gradually becomes lost during corneal storage. VIP treatment (10(-8) M, 37 °C, 30 min) prior to storage of freshly dissected human donor corneoscleral explants increases their CE cell CNTFRα level and responsiveness to CNTF in upregulating the gap junctional protein connexin-43 expression. VIP treatment of both fresh and preserved corneoscleral explants reduces CE damage in the corneoscleral explants and in the corneal buttons trephined from them. CE cell loss is a critical risk factor in corneal graft failure at any time in the life of the graft, which can be as late as 5-10 years after an initially successful transplant. A new procedure, Descemet's stripping automated endothelial keratoplasty (DSAEK), which is superior to the traditional full thickness transplantation in many aspects, nevertheless subjects the corneal endothelium to extensive mechanical forces, resulting in even more pronounced CE cell loss than the traditional technique. Whereas it is known that cells transduce mechanical stress through N-cadherin, stimulation of the N-cadherin pathway increases the anti-apoptotic protein Bcl-2 expression. Since N-cadherin and Bcl-2 in the corneal endothelium are both upregulated by VIP, we aim to strengthen the CE sheet by VIP treatments of the corneoscleral explants for full thickness traditional corneal transplantation and pre-cut corneas for DSAEK. Copyright © 2011 Elsevier Ltd. All rights reserved.

Expression of anti-Mullerian hormone in hens selected for different ovulation rates.

PubMed

Johnson, P A; Kent, T R; Urick, M E; Trevino, L S; Giles, J R

2009-05-01

In hens, the granulosa layer is the primary source of anti-Mullerian hormone (AMH), as it is in mammals. Small follicles express the greatest amount of Amh mRNA with less in the larger follicles. Laying hens have a distinct ovarian hierarchy of follicles while broiler breeder hens often have excessive follicle growth with a disrupted hierarchy. The objective of Experiment 1 was to examine Amh expression in two strains of hens differing in ovulatory efficiency. Amh expression was greater (P<0.01) in broiler breeder hens (n=6) as compared with laying hens (n=6). Experiment 2 was designed to examine whether alterations in follicular development due to diet, within the broiler breeder hens, were correlated with changes in the expression of Amh. Restricted feeding (RF) in broiler breeder hens promotes optimal follicular development. Egg production in broiler breeder hens on full feed (FF; n=8) was 78% that of hens on RF (n=9). The number of large follicles (P<0.05), total ovarian weight (P<0.01), and Amh mRNA expression were greater in FF hens as compared with RF hens (P<0.01). There was no difference in FSH receptor expression between the two groups. A direct nutritional effect was not supported because culture of granulosa cells with varying concentrations of glucose and insulin showed no effect on granulosa Amh expression. Finally, testis-conditioned medium resulted in a dose-related increase in granulosa cell proliferation, which could be inhibited by preincubation with AMH antibody. AMH may enhance granulosa cell proliferation through an autocrine or paracrine mechanism although excessive AMH may inhibit optimal follicle selection.

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

PubMed

Furtado, Milena B; Merriner, D Jo; Berger, Silke; Rhodes, Danielle; Jamsai, Duangporn; O'Bryan, Moira K

2017-12-01

The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described. Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations. Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

PubMed

Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

2013-10-01

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

The characteristics of vasa gene from Japanese sea bass ( Lateolabrax japonicas) and its response to the external hormones

NASA Astrophysics Data System (ADS)

Chi, Meili; Wen, Haishen; Ni, Meng; Qian, Kun; Zhang, Pei; Chai, Senhao

2015-08-01

The RNA helicase Vasa is an important regulator of primordial germ cell development. Its function in mature fish, especially the hormone-related differences in maturing male fish has seldom been documented. In this study, a full length cDNA sequence of the vasa gene was cloned from Japanese sea bass, Lateolabrax japonicas, and it was named jsb-vasa. Homology analysis showed that jsb-vasa was closely related to its teleost homologs. The spatial distribution of jsb-vasa indicated that it was only highly expressed in testis, showing its germ cell-specific expression pattern. During the testicular development cycle, jsb-vasa was highly expressed during early period of spermatogenesis, and reduced when spermatogenesis advanced. In addition, the jsb-vasa gene expression was significantly inhibited at 6 h, 12 h and 24 h after injecting hCG (human chorionic gonadotropin) and GnRHa (Gonadotropin-releasing hormone analogue), indicating that jsb-vasa gene may play an important role in spermatogenesis of Japanese sea bass, and be under the regulation of external sex hormones.

The human desmin locus: gene organization and LCR-mediated transcriptional control.

PubMed

Tam, Jennifer L Y; Triantaphyllopoulos, Kostas; Todd, Helen; Raguz, Selina; de Wit, Ton; Morgan, Jennifer E; Partridge, Terence A; Makrinou, Eleni; Grosveld, Frank; Antoniou, Michael

2006-06-01

Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between -9 and -18 kb 5' of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

PubMed

Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

2016-08-01

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

Kinin and Purine Signaling Contributes to Neuroblastoma Metastasis.

PubMed

Ulrich, Henning; Ratajczak, Mariusz Z; Schneider, Gabriela; Adinolfi, Elena; Orioli, Elisa; Ferrazoli, Enéas G; Glaser, Talita; Corrêa-Velloso, Juliana; Martins, Poliana C M; Coutinho, Fernanda; Santos, Ana P J; Pillat, Micheli M; Sack, Ulrich; Lameu, Claudiana

2018-01-01

Bone marrow metastasis occurs in approximately 350,000 patients that annually die in the U.S. alone. In view of the importance of tumor cell migration into the bone marrow, we have here investigated effects of various concentrations of stromal cell-derived factor-1 (SDF-1), bradykinin- and ATP on bone marrow metastasis. We show for first time that bradykinin augmented chemotactic responsiveness of neuroblastoma cells to SDF-1 and ATP concentrations, encountered under physiological conditions. Bradykinin upregulated VEGF expression, increased metalloproteinase activity and induced adhesion of neuroblastoma cells. Bradykinin augmented SDF-1-induced intracellular Ca 2+ mobilization as well as resensitization and expression of ATP-sensing P2X7 receptors. Bradykinin treatment resulted in higher gene expression levels of the truncated P2X7B receptor compared to those of the P2X7A full-length isoform. Bradykinin as pro-metastatic factor induced tumor proliferation that was significantly decreased by P2X7 receptor antagonists; however, the peptide did not enhance cell death nor P2X7A receptor-related pore activity, promoting neuroblastoma growth. Furthermore, immunodeficient nude/nude mice transplanted with bradykinin-pretreated neuroblastoma cells revealed significantly higher metastasis rates compared to animals injected with untreated cells. In contrast, animals receiving Brilliant Blue G, a P2X7 receptor antagonist, did not show any specific dissemination of neuroblastoma cells to the bone marrow and liver, and metastasis rates were drastically reduced. Our data suggests correlated actions of kinins and purines in neuroblastoma dissemination, providing novel avenues for clinic research in preventing metastasis.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

PubMed Central

Cherry, Jonathan J; Osman, Erkan Y; Evans, Matthew C; Choi, Sungwoon; Xing, Xuechao; Cuny, Gregory D; Glicksman, Marcie A; Lorson, Christian L; Androphy, Elliot J

2013-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full-length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high-throughput screening assay to identify small molecules that increase the expression of full-length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA. PMID:23740718

Molecular cloning and functional characterization of cathepsin D from sea cucumber Apostichopus japonicus.

PubMed

Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Qin, Lei; Du, Ming

2017-11-01

Cathepsin D (CTSD, EC 3.4.23.5) belongs to aspartic protease family, which is located in lysosomes and is distributed in diverse tissues and cells. CTSD has a wide variety of physiological functions, owing to its proteolytic activity in degradating proteins and peptides. In the current study, the full length cDNA of sea cucumber (Apostichopus japonicus) cathepsin D (AjCTSD) was firstly cloned, then the association between AjCTSD and sea cucumber autolysis was investigated. The full length cDNA of AjCTSD was 2896 bp, with an open reading frame (ORF) for 391 amino acids. AjCTSD was widely expressed in body wall, muscle and intestine; the expression level was the highest in intestine, followed by muscle and body wall. Compared to fresh tissues, AjCTSD expression levels were significantly increased in all examined autolytic tissues. The purified recombinant AjCTSD promoted the degradation of sea cucumber muscle. In conclusion, AjCTSD contributed to sea cucumber muscle autolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetic toxicology and toxicogenomic analysis of three cigarette smoke condensates in vitro reveals few differences among full-flavor, blonde, and light products

PubMed Central

Yauk, Carole L; Williams, Andrew; Buick, Julie K; Chen, Guosheng; Maertens, Rebecca M; Halappanavar, Sabina; White, Paul A

2012-01-01

Cigarette smoking leads to various detrimental health outcomes. Tobacco companies produce different brands of cigarettes that are marketed as reduced harm tobacco products. Early examples included “light” cigarettes, which differ from regular cigarettes due to filter ventilation and/or differences in chemical constituents. In order to establish baseline similarities and differences among different tobacco brands available in Canada, the present study examined the cytotoxicity, mutagenicity, clastogenicity, and gene expression profiles of cigarette smoke condensate (CSC) from three tobacco products, encompassing a full-flavor, blonde, and “light” variety. Using the Salmonella mutagenicity assay, we confirmed that the three CSCs are mutagenic, and that the potency is related to the presence of aromatic amines. Using the Muta™Mouse FE1 cell line we determined that the CSCs were clastogenic and cytotoxic, but nonmutagenic, and the results showed few differences in potencies among the three brands. There were no clear brand-specific changes in gene expression; each brand yielded highly similar expression profiles within a time point and concentration. The molecular pathways and biological functions affected by exposure included xenobiotic metabolism, oxidative stress, DNA damage response, cell cycle arrest and apoptosis, as well as inflammation. Thus, there was no appreciable difference in toxicity or gene expression profiles between regular brands and products marketed as “light,” and hence no evidence of reduced harm. The work establishes baseline CSC cytotoxicity, mutagenicity, and expression profiles that can be used as a point of reference for comparison with data generated for products marketed as reduced harm and/or modified risk tobacco products. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.† PMID:22431010

An efficient Foxtail mosaic virus vector system with reduced environmental risk

PubMed Central

2010-01-01

Background Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment. Results In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low Agrobacterium-mediated transformation rate of monocots. Conclusions The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing. PMID:21162736

Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells.

PubMed

Hlin, Hao; Zhi-Guo, Zhang; Cong-Hui, Han; Yan, Zhao; Qing, Liang; Bo, Jiang; Hou-Guang, He; Jun-Jie, Zhang; Pei-Ying, Zhang

2016-06-01

Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells. MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-β-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments. Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner. It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.

Canine distemper virus isolated from a monkey efficiently replicates on Vero cells expressing non-human primate SLAM receptors but not human SLAM receptor.

PubMed

Feng, Na; Liu, Yuxiu; Wang, Jianzhong; Xu, Weiwei; Li, Tiansong; Wang, Tiecheng; Wang, Lei; Yu, Yicong; Wang, Hualei; Zhao, Yongkun; Yang, Songtao; Gao, Yuwei; Hu, Guixue; Xia, Xianzhu

2016-08-02

In 2008, an outbreak of canine distemper virus (CDV) infection in monkeys was reported in China. We isolated CDV strain (subsequently named Monkey-BJ01-DV) from lung tissue obtained from a rhesus monkey that died in this outbreak. We evaluated the ability of this virus on Vero cells expressing SLAM receptors from dog, monkey and human origin, and analyzed the H gene of Monkey-BJ01-DV with other strains. The Monkey-BJ01-DV isolate replicated to the highest titer on Vero cells expressing dog-origin SLAM (10(5.2±0.2) TCID50/ml) and monkey-origin SLAM (10(5.4±0.1) TCID50/ml), but achieved markedly lower titers on human-origin SLAM cells (10(3.3±0.3) TCID50/ml). Phylogenetic analysis of the full-length H gene showed that Monkey-BJ01-DV was highly related to other CDV strains obtained during recent CDV epidemics among species of the Canidae family in China, and these Monkey strains CDV (Monkey-BJ01-DV, CYN07-dV, Monkey-KM-01) possessed a number of amino acid specific substitutions (E276V, Q392R, D435Y and I542F) compared to the H protein of CDV epidemic in other animals at the same period. Our results suggested that the monkey origin-CDV-H protein could possess specific substitutions to adapt to the new host. Monkey-BJ01-DV can efficiently use monkey- and dog-origin SLAM to infect and replicate in host cells, but further adaptation may be required for efficient replication in host cells expressing the human SLAM receptor.

Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles.

PubMed

Radeghieri, Annalisa; Savio, Giulia; Zendrini, Andrea; Di Noto, Giuseppe; Salvi, Alessandro; Bergese, Paolo; Piovani, Giovanna

2017-01-29

An increasing number of studies on stem cells suggests that the therapeutic effect they exert is primarily mediated by a paracrine regulation through extracellular vesicles (EVs) giving solid grounds for stem cell EVs to be exploited as agents for treating diseases or for restoring damaged tissues and organs. Due to their capacity to differentiate in all embryonic germ layers, amniotic fluid stem cells (AFCs), represent a highly promising cell type for tissue regeneration, which however is still poorly studied and in turn underutilized. In view of this, we conducted a first investigation on the expression of human hTERT gene - known to be among the key triggers of organ regeneration - in AFCs and in the EVs they secrete. Isolated AFCs were evaluated by RT-qPCR for hTERT expression. The clones expressing the highest levels of transcript, were analyzed by Immunofluorescence imaging and Nuclear/cytoplasmic fractionation in order to evaluate hTERT subcellular localization. We then separated EVs from FBS depleted culture medium by serial (ultra) centrifugations steps and characterized them using Western blotting, Atomic force Microscopy and Nanoplasmonic assay. We first demonstrated that primary cultures of AFCs express the gene hTERT at different levels. Then we evidenced that in AFCs with the higher transcript levels, the hTERT protein is present in the nuclear and cytoplasmic compartment. Finally, we found that cytosolic hTERT is embodied in the EVs that AFCs secrete in the extracellular milieu. Our study demonstrates for the first time the expression of the full protein hTERT by AFCs and its release outside the cell mediated by EVs, indicating a new extra telomeric role for this protein. This finding represents an initial but crucial evidence for considering AFCs derived EVs as new potential sources for tissue regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

PubMed Central

Lan, Feng; Liu, Junwei; Narsinh, Kazim H.; Hu, Shijun; Han, Leng; Lee, Andrew S.; Karow, Marisa; Nguyen, Patricia K.; Nag, Divya; Calos, Michele P.; Robbins, Robert C.; Wu, Joseph C.

2012-01-01

Background Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. Methods and Results We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. Conclusions The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types. PMID:22965984

CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth.

PubMed

Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A; Andl, Thomas; Zhang, Yuhang

2016-10-01

Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Human Myo19 is a novel myosin that associates with mitochondria

PubMed Central

Quintero, Omar A.; DiVito, Melinda M.; Adikes, Rebecca C.; Kortan, Melisa B.; Case, Lindsay B.; Lier, Audun J.; Panaretos, Niki S.; Slater, Stephanie Q.; Rengarajan, Michelle; Feliu, Marianela; Cheney, Richard E.

2009-01-01

Summary Mitochondria are pleomorphic organelles [1, 2] that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease [3-5]. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport [6]. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues and antibodies to human Myo19 detect a ∼109kD band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain-of-function where the majority of the mitochondria move continuously. Moving mitochondria travel for many microns with an obvious leading end and distorted shape. The motility and shape-change are sensitive to latrunculin B, indicating that both are actin-dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells. PMID:19932026

Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum

PubMed Central

Choi, Eunyoung; Roland, Joseph T.; Barlow, Brittney J.; O’Neal, Ryan; Rich, Amy E.; Nam, Ki Taek; Shi, Chanjuan; Goldenring, James R.

2014-01-01

Objective The glands of the stomach body and antral mucosa contain a complex compendium of cell lineages. In lower mammals, the distribution of oxyntic glands and antral glands define the anatomical regions within the stomach. We examined in detail the distribution of the full range of cell lineages within the human stomach. Design We determined the distribution of gastric gland cell lineages with specific immunocytochemical markers in entire stomach specimens from three non-obese organ donors. Results The anatomical body and antrum of the human stomach were defined by the presence of ghrelin and gastrin cells, respectively. Concentrations of somatostatin cells were observed in the proximal stomach. Parietal cells were seen in all glands of the body of stomach as well as in over 50% of antral glands. MIST1-expressing chief cells were predominantly observed in the body, although individual glands of the antrum also showed MIST1-expressing chief cells. While classically-described antral glands were observed with gastrin cells and deep antral mucous cells without any parietal cells, we also observed a substantial population of mixed-type glands containing both parietal cells and G cells throughout the antrum. Conclusions Enteroendocrine cells show distinct patterns of localization in the human stomach. The existence of antral glands with mixed cell lineages indicates that human antral glands may be functionally chimeric with glands assembled from multiple distinct stem cell populations. PMID:24488499

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

PubMed Central

Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

2009-01-01

Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Conclusions Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. PMID:19773264

Towards a Therapeutic Reduction of Imatinib Refractory Myeloproliferative Neoplasm-Initiating Cells

PubMed Central

Philips, Steven T.; Hildenbrand, Zacariah L.; Oravecz-Wilson, Katherine I.; Foley, S. Blake; Mgbemena, Victoria E.; Ross, Theodora S.

2014-01-01

Myeloproliferative neoplasms (MPNs) such as chronic myelogenous (CML) and chronic myelomonocytic leukemias (CMML) are frequently induced by tyrosine kinase oncogenes. Although these MPNs are sensitive to tyrosine kinase inhibitors such as imatinib, patients often relapse upon withdrawal of therapy. We used a model of MPN, which is induced by co-expression of the oncoproteins HIP1/PDGFβR (H/P) and AML1/ETO (A/E) from their endogenous loci, to examine the mechanisms of disease development and recurrence following imatinib withdrawal. Although the MPN displayed a full hematologic response to imatinib, 100% of the diseased mice relapsed upon drug withdrawal. MPN persistence was not due to imatinib resistance mutations in the H/P oncogene or massive gene expression changes. Within one week of imatinib treatment, more than 98% of gene expression changes induced by the oncogenes in isolated hematopoietic stem and progenitor cells (LSKs) normalized. Supplementation of imatinib with G-CSF or arsenic trioxide reduced MPN-initiating cell frequencies and the combination of imatinib with arsenic trioxide cured a large fraction of mice with MPNs. In contrast, no mice in the imatinib-treated control cohorts were cured. These data suggest that treatment with a combination of arsenic trioxide and imatinib can eliminate refractory MPN-initiating cells and reduce disease relapse. PMID:24240679

Toward a therapeutic reduction of imatinib refractory myeloproliferative neoplasm-initiating cells.

PubMed

Philips, S T; Hildenbrand, Z L; Oravecz-Wilson, K I; Foley, S B; Mgbemena, V E; Ross, T S

2014-11-13

Myeloproliferative neoplasms (MPNs) such as chronic myelogenous (CML) and chronic myelomonocytic leukemias (CMML) are frequently induced by tyrosine kinase oncogenes. Although these MPNs are sensitive to tyrosine kinase inhibitors such as imatinib, patients often relapse upon withdrawal of therapy. We used a model of MPN, which is induced by co-expression of the oncoproteins HIP1/PDGFβR (H/P) and AML1/ETO from their endogenous loci, to examine the mechanisms of disease development and recurrence following imatinib withdrawal. Although the MPN displayed a full hematologic response to imatinib, 100% of the diseased mice relapsed upon drug withdrawal. MPN persistence was not due to imatinib resistance mutations in the H/P oncogene or massive gene expression changes. Within 1 week of imatinib treatment, more than 98% of gene expression changes induced by the oncogenes in isolated hematopoietic stem and progenitor cells (lineage(-)Sca-1(+)c-Kit(+) immunophenotype) normalized. Supplementation of imatinib with granulocyte colony-stimulating factor or arsenic trioxide reduced MPN-initiating cell frequencies and the combination of imatinib with arsenic trioxide cured a large fraction of mice with MPNs. In contrast, no mice in the imatinib-treated control cohorts were cured. These data suggest that treatment with a combination of arsenic trioxide and imatinib can eliminate refractory MPN-initiating cells and reduce disease relapse.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang

Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a rolemore » in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.« less

Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

PubMed

Duncan, R; Horne, D; Strong, J E; Leone, G; Pon, R T; Yeung, M C; Lee, P W

1991-06-01

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression.

PubMed

Kinoshita, Rie; Watanabe, Masami; Huang, Peng; Li, Shun-Ai; Sakaguchi, Masakiyo; Kumon, Hiromi; Futami, Junichiro

2015-06-01

Reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3 is a tumor-suppressor gene and has been studied as a promising therapeutic gene for cancer gene therapy. Intratumoral injection of an adenovirus vector carrying the human REIC/Dkk-3 gene (Ad-REIC) elicits cancer cell-specific apoptosis and anticancer immune responses. The cytokine-like effect of secretory REIC/Dkk-3 on the induction of dendritic cell (DC)-like cell differentiation from monocytes plays a role in systemic anticancer immunity. In the present study, we generated recombinant full-length and N-terminally truncated REIC/Dkk-3 to characterize the biological activity of the protein. During the purification procedure, we identified a 17 kDa cysteine-rich stable product (C17-REIC) showing limited degradation. Further analysis showed that the C17-REIC domain was sufficient for the induction of DC-like cell differentiation from monocytes. Concomitant with the differentiation of DCs, the REIC/Dkk-3 protein induced the phosphorylation of glycogen synthase kinase 3β (GSK-3β) and signal transducers and activators of transcription (STAT) at a level comparable to that of granulocyte/macrophage colony-stimulating factor. In a mouse model of subcutaneous renal adenocarcinoma, intraperitoneal injection of full-length and C17-REIC proteins exerted anticancer effects in parallel with the activation of immunocompetent cells such as DCs and cytotoxic T lymphocytes in peripheral blood. Taken together, our results indicate that the stable cysteine-rich core region of REIC/Dkk-3 is responsible for the induction of anticancer immune responses. Because REIC/Dkk-3 is a naturally circulating serum protein, the upregulation REIC/Dkk-3 protein expression could be a promising option for cancer therapy.

Cell growth and migration under octenidine-antiseptic treatment.

PubMed

Jenull, S; Hojdar, K; Laggner, H; Velimirov, B; Zemann, N; Huettinger, M

2015-06-01

The toxicity of octenidine antiseptics in cultured cells contrasts their good tolerability in tissue. This phenomenon prompted us to examine which cell culture conditions allow survival and proliferation and to investigate a possible modulation of toxicity by the extracellular matrix proteoglycan chondroitin sulfate. We tested fibroblasts and MCF7 cells for growth using the MTT test, and assessed wound healing potency with a laceration assay. Expression levels of the genes involved in controlling wound healing were assessed with RT-PCR. A 24 hour exposure to the octenidine-based solution was found incompatible with cell growth. When octenidine solution (0.5-0.5mg/l) was coated on dishes, growth was profoundly reduced after 24 hours, however there was no cytotoxic effect at 0.012 mg/l. Interestingly, when dishes were first coated with chondroitin sulfate the cytotoxicity of octenidine-based solution was modulated. Cell migration was not inhibited by octenidine-based solution treatment (2 minutes; 15 mg/l). No significant changes in gene expression levels in response to the octenidine-based solution treatment were detected. In cell culture conditions application of the octenidine-based solution without toxicity can be observed, comparable to the minimal application required to give full bactericidal effect. Alteration of toxicity by interaction with chondroitin sulfate in cell culture suggests a similar function for extraceullar matrix in intact tissue.

Molecular Cloning and Characterization of Two Pig Vasoactive Intestinal Polypeptide Receptors (VPAC1-R and VPAC2-R)

PubMed Central

He, Xiaping; Meng, Fengyan; Wang, Yajun

2014-01-01

We here report the cloning, tissue expression, and functional analyses of the two pig vasoactive intestinal polypeptide (VIP) receptors (pVPAC1-R and pVPAC2-R). The cloned full-length pVPAC1-R and pVPAC2-R share high structural similarity with their mammalian counterparts. Functional assay revealed that the full-length pVPAC1-R and pVPAC2-R-expressed Chinese hamster ovary (CHO) cells could be activated by pVIP and pPACAP38 potently, indicating that pVPAC1-R and pVPAC2-R are capable of binding VIP and pituitary adenylate cyclase-activating polypeptide (PACAP). In addition to the identification of the transcripts encoding the two full-length receptors, multiple splice transcript variants were isolated. Comparison with the pig genome database revealed that pVPAC1-R and pVPAC2-R share a unique gene structure with 14 exons different from other vertebrates. Reverse transcription and polymerase chain reaction (RT-PCR) assays further showed that the transcript encoding the full-length pVPAC2-R is widely expressed in all adult tissues whereas the splice variants of pVPAC1-R are predominantly expressed in all tissues instead of the transcript encoding the full-length receptor, hinting that pVPAC2-R may play more important roles than pVPAC1-R in mediating VIP and PACAP actions. Our present findings help to elucidate the important role of VIP and PACAP and promote to rethink of their species-specific physiological roles including their actions in regulation of phenotypic traits in pigs. PMID:24520933

Tumor Progression Is Mediated by Thymosin-β4 through a TGFβ/MRTF Signaling Axis.

PubMed

Morita, Tsuyoshi; Hayashi, Ken'ichiro

2018-05-01

Although enhanced thymosin β4 (TMSB4X/Tβ4) expression is associated with tumor progression and metastasis, its tumor-promoting functions remain largely unknown. Here, it is demonstrated that TGFβ facilitates Tβ4 expression and leads to the activation of myocardin-related transcription factors (MRTF), which are coactivators of serum response factor (SRF) and regulate the expression of genes critical for the epithelial-mesenchymal transition (EMT) and tumor metastasis. In murine mammary gland cells (NMuMG), Tβ4 upregulation is required for full induction of a MRTF-regulated EMT gene expression program after TGFβ stimulation. Tβ4 levels are transcriptionally regulated via the novel cis -acting element AGACAAAG, which interacts with Smad and T-cell factor/lymphoid enhancer factor (TCF/LEF) to synergistically activate the Tβ4 promoter downstream of TGFβ. Murine skin melanoma cells (B16F0 and B16F1) also show the expression regulation of Tβ4 by Smad and TCF/LEF. Tβ4-knockout B16F1 (Tβ4 KO) clones show significantly diminished expression level of tumor-associated genes, which is regulated by the TGFβ/MRTFs pathway. In multiple human cancers, Tβ4 levels correlate positively with TGFβ1 and the tumor-associated gene expression levels through processes that respectively depend on TGFβ receptor 1 (TGFBR1) and MRTF expression. Kaplan-Meier survival analyses demonstrate that high Tβ4 expression associates with poor prognosis in an SRF expression-dependent manner in several cancers. In mice, Tβ4 KO clones show significantly decreased experimental metastatic potential; furthermore, ectopic expression of constitutively active MRTF-A fully restores the diminished metastatic activity. In conclusion, the TGFβ/Tβ4/MRTF/SRF pathway is critical for metastasis and tumor progression. Implications: These findings define a molecular mechanism underlying a tumor-promoting function of thymosin β4 through activation of MRTF/SRF signaling. Mol Cancer Res; 16(5); 880-93. ©2018 AACR . ©2018 American Association for Cancer Research.

Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells.

PubMed

Mellough, Carla B; Sernagor, Evelyne; Moreno-Gimeno, Inmaculada; Steel, David H W; Lako, Majlinda

2012-04-01

Recent successes in the stem cell field have identified some of the key chemical and biological cues which drive photoreceptor derivation from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC); however, the efficiency of this process is variable. We have designed a three-step photoreceptor differentiation protocol combining previously published methods that direct the differentiation of hESC and hiPSC toward a retinal lineage, which we further modified with additional supplements selected on the basis of reports from the eye field and retinal development. We report that hESC and hiPSC differentiating under our regimen over a 60 day period sequentially acquire markers associated with neural, retinal field, retinal pigmented epithelium and photoreceptor cells, including mature photoreceptor markers OPN1SW and RHODOPSIN with a higher efficiency than previously reported. In addition, we report the ability of hESC and hiPSC cultures to generate neural and retinal phenotypes under minimal culture conditions, which may be linked to their ability to endogenously upregulate the expression of a range of factors important for retinal cell type specification. However, cultures that were differentiated with full supplementation under our photoreceptor-induction regimen achieve this within a significantly shorter time frame and show a substantial increase in the expression of photoreceptor-specific markers in comparison to cultures differentiated under minimal conditions. Interestingly, cultures supplemented only with B27 and/or N2 displayed comparable differentiation efficiency to those under full supplementation, indicating a key role for B27 and N2 during the differentiation process. Furthermore, our data highlight an important role for Dkk1 and Noggin in enhancing the differentiation of hESC and hiPSC toward retinal progenitor cells and photoreceptor precursors during the early stages of differentiation, while suggesting that further maturation of these cells into photoreceptors may not require additional factors and can ensue under minimal culture conditions. Copyright © 2012 AlphaMed Press.

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

PubMed

Schanke, J T; Conwell, C M; Durning, M; Fisher, J M; Golos, T G

1997-03-01

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Mammalian cell display technology coupling with AID induced SHM in vitro: an ideal approach to the production of therapeutic antibodies.

PubMed

Qin, Chang-Fei; Li, Guan-Cheng

2014-12-01

Traditional antibody production technology within non-mammalian cell expression systems has shown many unsatisfactory properties for the development of therapeutic antibodies. Nevertheless, mammalian cell display technology reaps the benefits of producing full-length all human antibodies. Together with the developed cytidine deaminase induced in vitro somatic hypermutation technology, mammalian cell display technology provides the opportunity to produce high affinity antibodies that might be ideal for therapeutic application. This review was concentrated on the development of the mammalian cell display technology as well as the activation-induced cytidine deaminase induced in vitro somatic hypermutation technology and their applications for the production of therapeutic antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Orthotopic Implantation of Intact Tumor Tissue Leads to Metastasis of OCUM-2MD3 Human Gastric Cancer in Nude Mice Visualized in Real Time by Intravital Fluorescence Imaging.

PubMed

Murakami, Takashi; Zhang, Yong; Wang, Xiaoen; Hiroshima, Yukihiko; Kasashima, Hiroaki; Yashiro, Masakazu; Hirakawa, Kosei; Miwa, Atsushi; Kiyuna, Tasuku; Matsuyama, Ryusei; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Hoffman, Robert M

2016-05-01

Orthotopic (literally "correct place") implantation of cancer in nude mice has long been known to be superior to subcutaneous transplantation because the orthotopic tumor can metastasize. We reported previously on surgical orthotopic implantation (SOI) of gastric cancer tissue in nude mice resulting in the formation of metastases in 100% of the mice with extensive primary growth to the regional lymph nodes, liver, and lung. In contrast, when cell suspensions were used to inject gastric cancer cells orthotopically, metastases occurred in only 6.7% of the mice with local tumor formation, emphasizing the importance of orthotopically implanting intact tissue to allow full expression of metastatic potential. However, the different behavior of tumors implanted orthotopically by the two methods has not been visualized in real time. OCUM-2MD3 human gastric cancer cells labeled with the fluorescent protein Azami-Green were implanted orthotopically as cells or tissue in nude mice. Orthotopic implantation of cells resulted in local spread on the stomach. In contrast, SOI of tumor tissue of OCUM-2MD3 resulted in vessel spread of the Azami-Green-expressing cancer cells. Metastasis was also observed in the left lobe of the liver after SOI. These results demonstrate the physiological importance of intact cancer tissue for orthotopic implantation in order for tumors to properly grow and express their metastatic potential. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

PubMed

Cotton, R N; McDonald-Fleming, R; Boyd, A; Spates, K; Nutman, T B; Tolouei Semnani, R

2015-02-01

Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin. © 2014 John Wiley & Sons Ltd.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

PubMed

Tyrrell, Christine; De Cecco, Martin; Reynolds, Natalie L; Kilanowski, Fiona; Campopiano, Dominic; Barran, Perdita; Macmillan, Derek; Dorin, Julia R

2010-03-01

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis. Copyright 2009 Elsevier Ltd. All rights reserved.

Bidirectional Modulation of Adipogenesis by the Secreted Protein Ccdc80/DRO1/URB*

PubMed Central

Tremblay, Frédéric; Revett, Tracy; Huard, Christine; Zhang, Ying; Tobin, James F.; Martinez, Robert V.; Gimeno, Ruth E.

2009-01-01

Adipocyte-secreted proteins play important roles in metabolic regulation through autocrine, paracrine, and endocrine mechanisms. Using transcriptional profiling, we identified coiled-coil domain containing 80 (Ccdc80; also known as DRO1 and URB) as a novel secreted protein highly expressed in white adipose tissue. In 3T3-L1 cells Ccdc80 is expressed and secreted in a biphasic manner with high levels in postconfluent preadipocytes and terminally differentiated adipocytes. To determine whether Ccdc80 regulates adipocyte differentiation, Ccdc80 expression was manipulated using both knockdown and overexpression approaches. Small hairpin RNA-mediated silencing of Ccdc80 in 3T3-L1 cells inhibits adipocyte differentiation. This phenotype was partially reversed by treating the knockdown cells with Ccdc80-containing conditioned medium from differentiated 3T3-L1 cells. Molecular studies indicate that Ccdc80 is required for the full inhibition of T-cell factor-mediated transcriptional activity, down-regulation of Wnt/β-catenin target genes during clonal expansion, and the subsequent induction of C/EBPα and peroxisome proliferator-activated receptor γ. Surprisingly, overexpression of Ccdc80 in 3T3-L1 cells also inhibits adipocyte differentiation without affecting the repression of the Wnt/β-catenin signaling pathway. Taken together, these data suggest that Ccdc80 plays dual roles in adipogenesis by mechanisms that involve at least in part down-regulation of Wnt/β-catenin signaling and induction of C/EBPα and peroxisome proliferator-activated receptor γ. PMID:19141617

Cysteine Dioxygenase 1 Is a Tumor Suppressor Gene Silenced by Promoter Methylation in Multiple Human Cancers

PubMed Central

Brait, Mariana; Ling, Shizhang; Nagpal, Jatin K.; Chang, Xiaofei; Park, Hannah Lui; Lee, Juna; Okamura, Jun; Yamashita, Keishi; Sidransky, David; Kim, Myoung Sook

2012-01-01

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer. PMID:23028699

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

PubMed

Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

2010-07-01

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

A Novel Terminal-Repeat Retrotransposon in Miniature (TRIM) Is Massively Expressed in Echinococcus multilocularis Stem Cells

PubMed Central

Koziol, Uriel; Radio, Santiago; Smircich, Pablo; Zarowiecki, Magdalena; Fernández, Cecilia; Brehm, Klaus

2015-01-01

Taeniid cestodes (including the human parasites Echinococcus spp. and Taenia solium) have very few mobile genetic elements (MGEs) in their genome, despite lacking a canonical PIWI pathway. The MGEs of these parasites are virtually unexplored, and nothing is known about their expression and silencing. In this work, we report the discovery of a novel family of small nonautonomous long terminal repeat retrotransposons (also known as terminal-repeat retrotransposons in miniature, TRIMs) which we have named ta-TRIM (taeniid TRIM). ta-TRIMs are only the second family of TRIM elements discovered in animals, and are likely the result of convergent reductive evolution in different taxonomic groups. These elements originated at the base of the taeniid tree and have expanded during taeniid diversification, including after the divergence of closely related species such as Echinococcus multilocularis and Echinococcus granulosus. They are massively expressed in larval stages, from a small proportion of full-length copies and from isolated terminal repeats that show transcriptional read-through into downstream regions, generating novel noncoding RNAs and transcriptional fusions to coding genes. In E. multilocularis, ta-TRIMs are specifically expressed in the germinative cells (the somatic stem cells) during asexual reproduction of metacestode larvae. This would provide a developmental mechanism for insertion of ta-TRIMs into cells that will eventually generate the adult germ line. Future studies of active and inactive ta-TRIM elements could give the first clues on MGE silencing mechanisms in cestodes. PMID:26133390

An electrospun scaffold integrating nucleic acid delivery for treatment of full thickness wounds

PubMed Central

Kobsa, Serge; Kristofik, Nina J.; Sawyer, Andrew J.; Bothwell, Alfred L.M.; Kyriakides, Themis R.; Saltzman, W. Mark

2013-01-01

We developed a multi-functional construct capable of controlled delivery of bioactive substances that can improve wound repair by supporting the intrinsic ability of the skin to heal. We synthesized electrospun scaffolds—composed of a blend of the degradable polymers poly(L-lactide) (PLA) or polycaprolactone (PCL)—that produce highly efficient non-viral in vivo gene delivery to cells in the wound bed, provide a protective barrier during early wound healing, and support cell migration and growth. This multi-functional material was tested for its influence on wound healing: scaffolds were loaded with plasmids encoding keratinocyte growth factor (KGF) and applied to full thickness wounds in mice. Compared to scaffolds with control plasmids, animals receiving the KGF plasmid-loaded scaffold produced significant enhancements in wound healing, which was quantified by improvements in the rate of wound re-epithelialization, keratinocyte proliferation, and granulation response. Further, we quantified the expression level of endogenous and plasmid-derived KGF in wound samples: qRT-PCR on wound sections revealed a correlation between the levels of plasmid-derived protein expression and histological analysis of wound healing, revealing an inverse relationship between the expression level of exogenous KGF and the size of the unhealed epithelial layer in wounds. Our findings suggest that engineered nanofiber PLA/PCL scaffolds are capable of highly efficient controlled DNA delivery and are promising materials for treatment of cutaneous wounds. PMID:23453058

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

PubMed

Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

2017-08-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer

PubMed Central

Hout, David R.; Lawrence, Kasey; Morris, Stephan W.; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly

2017-01-01

Patients with lung cancers harboring an activating anaplastic lymphoma kinase (ALK) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off ΔCt of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK. PMID:28763012

Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner

PubMed Central

Johnson, Rachelle W.; Sun, Yao; Ho, Patricia W. M.; Chan, Audrey S. M.; Johnson, Jasmine A.; Pavlos, Nathan J.; Sims, Natalie A.; Martin, T. John

2018-01-01

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells “home” to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1–141) and PTH(1–34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule. PMID:29867773

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were asmore » capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.« less

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

PubMed Central

Giuliani, Anna Lisa; Colognesi, Davide; Ricco, Tiziana; Roncato, Carlotta; Capece, Marina; Amoroso, Francesca; Wang, Qi Guang; De Marchi, Elena; Gartland, Allison; Di Virgilio, Francesco; Adinolfi, Elena

2014-01-01

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca2+ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity. PMID:25226385

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

NASA Astrophysics Data System (ADS)

Saren, Gaowa; Zhao, Yonggang

2009-05-01

A full length amphioxus cDNA, encoding a novel phosducin-like protein ( Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/ Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

PubMed Central

Chan, Kok-Fei; Oveissi, Sara; Jackson, Heather M.; Dimopoulos, Nektaria; Guillaume, Philippe; Knights, Ashley J.; Lowen, Tamara; Robson, Neil C.; Russell, Sarah E.; Scotet, Emmanuel; Davis, Ian D.; Maraskovsky, Eugene; Cebon, Jonathan; Luescher, Immanuel F.; Chen, Weisan

2012-01-01

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. PMID:22970293

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function.

PubMed

Hemingway, Cheryl; Berk, Maurice; Anderson, Suzanne T; Wright, Victoria J; Hamilton, Shea; Eleftherohorinou, Hariklia; Kaforou, Myrsini; Goldgof, Greg M; Hickman, Katy; Kampmann, Beate; Schoeman, Johan; Eley, Brian; Beatty, David; Pienaar, Sandra; Nicol, Mark P; Griffiths, Michael J; Waddell, Simon J; Newton, Sandra M; Coin, Lachlan J; Relman, David A; Montana, Giovanni; Levin, Michael

2017-01-01

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.

Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

PubMed Central

Hendaoui, Ismaïl; Lavergne, Elise; Lee, Heun-Sik; Hong, Seong Hyun; Kim, Hak-Zoo; Parent, Christelle; Heuzé-Vourc'h, Nathalie; Clément, Bruno; Musso, Orlando

2012-01-01

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth. PMID:22303445

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

PubMed

Cattaruzza, Sabrina; Nicolosi, Pier Andrea; Braghetta, Paola; Pazzaglia, Laura; Benassi, Maria Serena; Picci, Piero; Lacrima, Katia; Zanocco, Daniela; Rizzo, Erika; Stallcup, William B; Colombatti, Alfonso; Perris, Roberto

2013-06-01

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

Activation of the N-Ras-PI3K-Akt-mTOR Pathway by Hepatitis C Virus: Control of Cell Survival and Viral Replication

PubMed Central

Mannová, Petra; Beretta, Laura

2005-01-01

The hepatitis C virus (HCV) replication complex is localized within detergent-resistant membranes or lipid rafts. We analyzed the protein contents of detergent-resistant fractions isolated from Huh7 cells expressing a self-replicating full-length HCV-1b genome. Using two-dimensional gel electrophoresis followed by mass spectrometry, we identified N-Ras as one of the proteins in which expression was increased in the detergent-resistant fractions from HCV genomic replicon clones compared to control cells. N-Ras is an activator of the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. We found that the activities of PI3K and Akt, as well as the activity of their downstream target, mTOR, in the HCV-replicating cells were increased. Both PI3K-Akt- and mTOR-dependent pathways have been shown to promote cell survival. In agreement with this, HCV replicon cells were resistant to serum starvation-induced apoptosis. We also characterized the role of this pathway in HCV replication. Reduction of N-Ras expression by transfection of N-Ras small interfering RNA (siRNA) resulted in increased replication of HCV. We observed a similar increase in HCV replication in cells treated with the PI3K inhibitor LY294002 and in cells transfected with mTOR siRNA. Taken together, these data suggest that increased N-Ras levels in subcellular sites of HCV replication and stimulation of the prosurvival PI3K-Akt pathway and mTOR by HCV not only protect cells against apoptosis but also contribute to the maintenance of steady-state levels of HCV replication. These effects may contribute to the establishment of persistent infection by HCV. PMID:15994768

Expression of the ctenophore Brain Factor 1 forkhead gene ortholog (ctenoBF-1) mRNA is restricted to the presumptive mouth and feeding apparatus: implications for axial organization in the Metazoa

NASA Technical Reports Server (NTRS)

Yamada, Atsuko; Martindale, Mark Q.

2002-01-01

Ctenophores are thoroughly modern animals whose ancestors are derived from a separate evolutionary branch than that of other eumetazoans. Their major longitudinal body axis is the oral-aboral axis. An apical sense organ, called the apical organ, is located at the aboral pole and contains a highly innervated statocyst and photodetecting cells. The apical organ integrates sensory information and controls the locomotory apparatus of ctenophores, the eight longitudinal rows of ctene/comb plates. In an effort to understand the developmental and evolutionary organization of axial properties of ctenophores we have isolated a forkhead gene from the Brain Factor 1 (BF-1) family. This gene, ctenoBF-1, is the first full-length nuclear gene reported from ctenophores. This makes ctenophores the most basal metazoan (to date) known to express definitive forkhead class transcription factors. Orthologs of BF-1 in vertebrates, Drosophila, and Caenorhabditis elegans are expressed in anterior neural structures. Surprisingly, in situ hybridizations with ctenoBF-1 antisense riboprobes show that this gene is not expressed in the apical organ of ctenophores. CtenoBF-1 is expressed prior to first cleavage. Transcripts become localized to the aboral pole by the 8-cell stage and are inherited by ectodermal micromeres generated from this region at the 16- and 32-cell stages. Expression in subsets of these cells persists and is seen around the edge of the blastopore (presumptive mouth) and in distinct ectodermal regions along the tentacular poles. Following gastrulation, stomodeal expression begins to fade and intense staining becomes restricted to two distinct domains in each tentacular feeding apparatus. We suggest that the apical organ is not homologous to the brain of bilaterians but that the oral pole of ctenophores corresponds to the anterior pole of bilaterian animals.

ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

PubMed

Lopez, M; Oettgen, P; Akbarali, Y; Dendorfer, U; Libermann, T A

1994-05-01

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells

PubMed Central

Uzbekova, Svetlana; Roy-Sabau, Monica; Dalbiès-Tran, Rozenn; Perreau, Christine; Papillier, Pascal; Mompart, Florence; Thelie, Aurore; Pennetier, Sophie; Cognie, Juliette; Cadoret, Veronique; Royere, Dominique; Monget, Philippe; Mermillod, Pascal

2006-01-01

Background Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. Methods Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. Results We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. Conclusion Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes. PMID:16551357

Blocking the expression of both bone sialoprotein (BSP) and osteopontin (OPN) impairs the anabolic action of PTH in mouse calvaria bone.

PubMed

Bouleftour, Wafa; Bouet, Guenaelle; Granito, Renata Neves; Thomas, Mireille; Linossier, Marie-Thérèse; Vanden-Bossche, Arnaud; Aubin, Jane E; Lafage-Proust, Marie-Hélène; Vico, Laurence; Malaval, Luc

2015-03-01

Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1-84 (iPTH) over the calvaria of BSP knockout (-/-) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and -/- mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from -/- mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in -/- versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in -/- cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in -/- mice, reduced to a modest increase in periosteum thickness. In -/- (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. © 2014 Wiley Periodicals, Inc., A Wiley Company.

Transection of Preganglionic Axons Leads to CNS Neuronal Plasticity Followed by Survival and Target Reinnervation

PubMed Central

Coulibaly, Aminata P.; Gannon, Sean M.; Hawk, Kiel; Walsh, Brian F.; Isaacson, Lori G.

2013-01-01

The goals of the present study were to investigate the changes in sympathetic preganglionic neurons following transection of distal axons in the cervical sympathetic trunk (CST) that innervate the superior cervical ganglion (SCG) and to assess changes in the protein expression of brain derived neurotrophic factor (BDNF) and its receptor TrkB in the thoracic spinal cord. . At 1 week, a significant decrease in soma volume and reduced soma expression of choline acetyltransferase (ChAT) in the intermediolateral cell column (IML) of T1 spinal cord were observed, with both ChAT-ir and non-immunoreactive neurons expressing the injury marker activating transcription factor 3. . These changes were transient, and at later time points, ChAT expression and soma volume returned to control values and the number of ATF3 neurons declined. No evidence for cell loss or neuronal apoptosis was detected at any time point. Protein levels of BDNF and/or full length TrkB in the spinal cord were increased throughout the survival period. In the SCG, both ChAT-ir axons and ChAT protein remained decreased at 16 weeks, but were increased compared to the 10 week time point. These results suggest that though IML neurons show reduced ChAT expression and cell volume at 1 week following CST transection, at later time points, the neurons recovered and exhibited no significant signs of neurodegeneration. The alterations in BDNF and/or TrkB may have contributed to the survival of the IML neurons and the recovery of ChAT expression, as well as to the reinnervation of the SCG. PMID:23891533

Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage*

PubMed Central

Gudi, Radhika; Haycraft, Courtney J.; Bell, P. Darwin; Li, Zihai; Vasu, Chenthamarakshan

2015-01-01

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis. PMID:25616662

Adapting in vitro embryonic stem cell differentiation to the study of locus control regions.

PubMed

Lahiji, Armin; Kučerová-Levisohn, Martina; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D

2014-05-01

Numerous locus control region (LCR) activities have been discovered in gene loci important to immune cell development and function. LCRs are a distinct class of cis-acting gene regulatory elements that appear to contain all the DNA sequence information required to establish an independently and predictably regulated gene expression program at any genomic site in native chromatin of a whole animal. As such, LCR-regulated transgenic reporter systems provide invaluable opportunities to investigate the mechanisms of gene regulatory DNA action during development. Furthermore the qualities of LCR-driven gene expression, including spatiotemporal specificity and "integration site-independence" would be highly desirable to incorporate into vectors used in therapeutic genetic engineering. Thus, advancement in the methods used to investigate LCRs is of considerable basic and translational significance. We study the LCR present in the mouse T cell receptor (TCR)-α gene locus. Until recently, transgenic mice provided the only experimental model capable of supporting the entire spectrum of LCR activities. We have recently reported complete manifestation of TCRα LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC), thus validating a complete cell culture model for the full range of LCR activities seen in transgenic mice. Here we discuss the critical parameters involved in studying LCR-regulated gene expression during in vitro hematopoietic differentiation from ESCs. This advance provides an approach to speed progress in the LCR field, and facilitate the clinical application of its findings, particularly to the genetic engineering of T cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

PubMed

Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis

PubMed Central

Subramanian, M; Francis, P; Bilke, S; Li, XL; Hara, T; Lu, X; Jones, MF; Walker, RL; Zhu, Y; Pineda, M; Lee, C; Varanasi, L; Yang, Y; Martinez, LA; Luo, J; Ambs, S; Sharma, S; Wakefield, LM; Meltzer, PS; Lal, A

2015-01-01

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis. PMID:24662829

Regulation of adipolin/CTRP12 cleavage by obesity.

PubMed

Enomoto, Takashi; Shibata, Rei; Ohashi, Koji; Kambara, Takahiro; Kataoka, Yoshiyuki; Uemura, Yusuke; Yuasa, Daisuke; Murohara, Toyoaki; Ouchi, Noriyuki

2012-11-09

Obesity is highly associated with the development of insulin resistance and type 2 diabetes. Recently we found that adipolin/CRTP12 is an adipocytokine that exerts beneficial actions on glucose metabolism. Here we investigated the regulation of circulating adipolin under conditions of obesity and assessed its potential mechanisms. Both full and cleaved forms of adipolin were observed in mouse plasma. Diet-induced obese (DIO) mice showed a significant reduction of plasma levels of full and total (full and cleaved) adipolin compared with control mice, resulting in an increase in the ratio of cleaved to full isoform. In vitro gene transfection studies using HEK293 cells revealed that a deletion mutant of adipolin gene (Δaa90-93) caused a reduction of cleaved production of adipolin in media. A bioinformatics analysis of adipolin amino acid sequence indicated the potential involvement of the family of proprotein convertases (PCs) in cleavage of adipolin. Treatment of 3T3-L1 adipocytes with an inhibitor for PCs abolished the expression of cleaved adipolin form in the media. The expression of furin, the member of PCs, was increased in adipose tissue of DIO mice. Furin expression was also increased in cultured adipocytes by treatment with an inducer of inflammation. These data suggest that obesity states facilitate the cleavage of adipolin presumably through upregulation of furin in adipose tissue. Copyright © 2012 Elsevier Inc. All rights reserved.

Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis.

PubMed

Klissenbauer, Monika; Winters, Silke; Heinlein, Uwe A O; Lisowsky, Thomas

2002-07-01

In this study, we investigated the expression of the mammalian FAD-dependent sulphydryl oxidase Erv1p/Alrp in the rat and mouse and during mouse spermatogenesis. Up to three forms of Alrp were identified in protein extracts from different tissues and organs, but very little enzyme was present in blood samples. The three forms of Alrp represent the full-length protein of 23 kDa and fragments of 21 kDa and 15 kDa. All forms of Alrp were assembled into dimers in vivo. In contrast to samples from other organs, the protein analysis of mouse testis identified predominantly full-length 23 kDa Alrp. This finding prompted us to investigate in more detail the expression of Alrp during spermatogenesis. Testis samples of individual mice from postnatal days 13-29 were probed with an antibody specific for mammalian Alrp. In addition, cells from whole testis preparations were fractionated on a bovine serum albumin column gradient. Protein expression of mouse Alrp was compared with those of testis-specific cyritestin, the cytoskeleton marker actin and mitochondrial subunit Vb of cytochrome oxidase and cytochrome c. These studies demonstrated a specific accumulation of full-length mouse Alrp during the early stages of spermatogenesis. The highest levels of Alrp were found in spermatogonia and primary spermatocytes. Levels of expression of Alrp did not correlate with the synthesis of components of the respiratory chain, indicating that full-length Alrp in the mitochondria of spermatogonia and spermatocytes has another function in addition to its role in oxidative phosphorylation.

Expression in Escherichia coli, refolding and crystallization of Aspergillus niger feruloyl esterase A using a serial factorial approach.

PubMed

Benoit, Isabelle; Coutard, Bruno; Oubelaid, Rachid; Asther, Marcel; Bignon, Christophe

2007-09-01

Hydrolysis of plant biomass is achieved by the combined action of enzymes secreted by microorganisms and directed against the backbone and the side chains of plant cell wall polysaccharides. Among side chains degrading enzymes, the feruloyl esterase A (FAEA) specifically removes feruloyl residues. Thus, FAEA has potential applications in a wide range of industrial processes such as paper bleaching or bio-ethanol production. To gain insight into FAEA hydrolysis activity, we solved its crystal structure. In this paper, we report how the use of four consecutive factorial approaches (two incomplete factorials, one sparse matrix, and one full factorial) allowed expressing in Escherichia coli, refolding and then crystallizing Aspergillus niger FAEA in 6 weeks. Culture conditions providing the highest expression level were determined using an incomplete factorial approach made of 12 combinations of four E. coli strains, three culture media and three temperatures (full factorial: 36 combinations). Aspergillus niger FAEA was expressed in the form of inclusion bodies. These were dissolved using a chaotropic agent, and the protein was purified by affinity chromatography on Ni column under denaturing conditions. A suitable buffer for refolding the protein eluted from the Ni column was found using a second incomplete factorial approach made of 96 buffers (full factorial: 3840 combinations). After refolding, the enzyme was further purified by gel filtration, and then crystallized following a standard protocol: initial crystallization conditions were found using commercial crystallization screens based on a sparse matrix. Crystals were then optimized using a full factorial screen.

Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

PubMed Central

Swevers, Luc; Liu, Jisheng; Huvenne, Hanneke; Smagghe, Guy

2011-01-01

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed. PMID:21637842

Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

PubMed

Fatima, Sarwat; Chui, Chung H; Tang, Wing K; Hui, Kin S; Au, Ho W; Li, Wing Y; Wong, Mei M; Cheung, Filly; Tsao, S W; Lam, King Y; Beh, Philip S L; Wong, John; Law, Simon; Srivastava, Gopesh; Ho, Kwok P; Chan, Albert S C; Tang, Johnny C O

2006-01-01

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Hayden, Patrick J.; Casillas, Robert P.

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT{sup TM}, a commercially available full-thickness human skin equivalent. CEES (100-1000 {mu}M) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 {mu}M), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histonemore » H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE{sub 2} synthases, leukotriene (LT) A{sub 4} hydrolase and LTC{sub 4} synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.« less

Fine mapping of canine parvovirus B cell epitopes.

PubMed

López de Turiso, J A; Cortés, E; Ranz, A; García, J; Sanz, A; Vela, C; Casal, J I

1991-10-01

In this report we describe the topological mapping of neutralizing domains of canine parvovirus (CPV). We obtained 11 CPV-specific monoclonal antibodies (MAbs), six of which are neutralizing. The reactivities were as determined by ELISA and Western blot (immunoblot) analysis. VP2, the most abundant protein of the CPV capsid, seemed to contain all the neutralization sites. Also, an almost full-length genomic clone of CPV was constructed in the bacterial plasmid pUC18 to enable expression of CPV proteins. All the neutralizing MAbs recognized recombinant VP2 when it was expressed as a free protein in Escherichia coli but not when expressed as a fusion protein with glutathione-S-transferase. When two large fragments containing about 85% and 67% of the C terminus of VP2 were expressed, no neutralization sites were detected. When fusion proteins containing the N terminus were expressed, two linear determinants were mapped, one between residues 1 to 10 of VP2, and the other between amino acids 11 and 23. The peptide 11 GQPAVRNERATGS 23, recognized by MAb 3C9, was synthesized chemically and checked for immunogenicity, not being able to induce neutralizing activity. Although the antibody response in rabbits to all the fusion proteins was uniformly high, the anti-CPV response was very variable. Protein from pCPVEx11, which contains a T cell epitope (peptide PKIFINLAKKKKAG) present in the VP1-specific region as well as the B cell epitopes, seemed to be the most effective in inducing virus neutralization.

Icaritin inhibits the expression of alpha-fetoprotein in hepatitis B virus-infected hepatoma cell lines through post-transcriptional regulation.

PubMed

Zhang, Chao; Li, Hui; Jiang, Wei; Zhang, Xiaowei; Li, Gang

2016-12-13

Although it has showed that icaritin can apparently suppress growth of HCC by reducing the level of AFP, the intrinsic mechanism remains unclear. In this study, we explored the possible mechanism of miRNAs on post-transcriptional regulation of AFP gene, as well as the effects of HBV infection and icaritin in hepatoma cells. The results showed that miR-620, miR-1236 and miR-1270 could bind target sites in the range of 9-18 nt and 131-151 nt downstream of the stop codon in the AFP mRNA 3'-UTR to suppress the expression of AFP. Mutation of these target sites could reverse the effects of these miRNAs. Icaritin (10 μM) might reduce the stability and translational activity of AFP mRNA by increasing the expression levels of these mentioned miRNAs. HBV infection resulted in apparent decreases of these miRNAs and, consequently, increased AFP expression. The results indicated that miR-620, miR-1236 and miR-1270 are critical factors in the post-transcriptional regulation of AFP. Icaritin can counteract the effect of HBV. These findings will contribute to full understanding of the regulatory mechanism of AFP expression in hepatoma cells. And also it revealed a synergistic mechanism of HBV infection and elevation of AFP in the pathogenesis of HCC, as well as the potential clinical significance of icaritin on the therapy of HCC induced by HBV.

Mesenchymal stem cells increase skin graft survival time and up-regulate PD-L1 expression in splenocytes of mice.

PubMed

Moravej, Ali; Geramizadeh, Bita; Azarpira, Negar; Zarnani, Amir-Hassan; Yaghobi, Ramin; Kalani, Mehdi; Khosravi, Maryam; Kouhpayeh, Amin; Karimi, Mohammad-Hossein

2017-02-01

Recently, mesenchymal stem cells (MSCs) have gained considerable interests as hopeful therapeutic cells in transplantation due to their immunoregulatory functions. But exact mechanisms underlying MSCs immunoregulatory function is not fully understood. Herein, in addition to investigate the ability of MSCs to prolong graft survival time, the effects of them on the expression of PD-L1 and IDO immunomodulatory molecules in splenocytes of skin graft recipient mice was clarified. To achieve this goal, full-thickness skins were transplanted from C57BL/6 to BALB/c mice. MSCs were isolated from bone marrow of BALB/c mice and injected to the recipient mice. Skin graft survival was monitored daily to determine graft rejection time. On days 2, 5 and 10 post skin transplantation, serum cytokine levels and expression of PD-L1 and IDO mRNA and protein in the splenocytes of recipient mice were evaluated. The results showed that administration of MSCs prolonged skin graft survival time from 11 to 14 days. On days 2 and 5 post transplantation, splenocytes PD-L1 expression and IL-10 serum level in MSCs treated mice were higher than those in the controls, while IL-2 and IFN-γ levels were lower. Rejection in MSCs treated mice was accompanied by an increase in IL-2 and IFN-γ, and decrease in PD-L1 expression and IL-10 level. No difference in the expression of IDO between MSCs treated mice and controls was observed. In conclusion, we found that one of the mechanisms underlying MSCs immunomodulatory function could be up-regulating PD-L1 expression. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro

PubMed Central

Yang, Dong-Ye; Lu, Fang-Gen; Tang, Xi-Xiang; Zhao, Shui-Ping; Ouyang, Chun-Hui; Wu, Xiao-Ping; Liu, Xiao-Wei; Wu, Xiao-Ying

2003-01-01

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P40 and P35 subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1 (±) to construct plasmids of P (+)/IL-12, P (+)/P40 and P (-)/P35. These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P (+)/IL-12 and ASOR-PLL-P (+)/P40 + ASOR-PLL-P (-)/P35] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P (+)/P40 and ASOR-PLL-P (-)/P35 was higher than that of ASOR-PLL-P (+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level. PMID:12970883

Fibrin gel as a scaffold for skin substitute – production and clinical experience.

PubMed

Kljenak, Antun; Tominac Trcin, Mirna; Bujić, Marina; Dolenec, Tamara; Jevak, Martina; Mršić, Gordan; Zmiš, Gordana; Barčot, Zoran; Muljačić, Ante; Popović, Maja

2016-06-01

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaff old. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.

Mouse RC/BTB2, a Member of the RCC1 Superfamily, Localizes to Spermatid Acrosomal Vesicles

PubMed Central

Shen, Xuening; Nagarkatti-Gude, David R.; Hess, Rex A.; Henderson, Scott C.; Strauss, Jerome F.; Zhang, Zhibing

2012-01-01

Mouse RC/BTB2 is an unstudied protein of the RCC1 (Regulator of Chromosome Condensation) superfamily. Because of the significant remodeling of chromatin that occurs during spermiogenesis, we characterized the expression and localization of mouse RC/BTB2 in the testis and male germ cells. The Rc/btb2 gene yields two major transcripts: 2.3 kb Rc/btb2-s, present in most somatic tissues examined; and 2.5 kb Rc/btb2-t, which contains a unique non-translated exon in its 5′-UTR that is only detected in the testis. During the first wave of spermatogenesis, Rc/btb2-t mRNA is expressed from day 8 after birth, reaching highest levels of expression at day 30 after birth. The full-length protein contains three RCC1 domains in the N-terminus, and a BTB domain in the C-terminus. In the testis, the protein is detectable from day 12, but is progressively up-regulated to day 30 and day 42 after birth. In spermatids, some of the protein co-localizes with acrosomal markers sp56 and peanut lectin, indicating that it is an acrosomal protein. A GFP-tagged RCC1 domain is present throughout the cytoplasm of transfected CHO cells. However, both GFP-tagged, full-length RC/BTB2 and a GFP-tagged BTB domain localize to vesicles in close proximity to the nuclear membrane, suggesting that the BTB domain might play a role in mediating full-length RC/BTB2 localization. Since RCC1 domains associate with Ran, a small GTPase that regulates molecular trafficking, it is possible that RC/BTB2 plays a role in transporting proteins during acrosome formation. PMID:22768142

Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein*

PubMed Central

Zhang, Xiaohong; He, Xiao; Baker, Joseph; Tama, Florence; Chang, Geoffrey; Wright, Stephen H.

2012-01-01

The x-ray structure of the prototypic MATE family member, NorM from Vibrio cholerae, reveals a protein fold composed of 12 transmembrane helices (TMHs), confirming hydropathy analyses of the majority of (prokaryotic and plant) MATE transporters. However, the mammalian MATEs are generally predicted to have a 13th TMH and an extracellular C terminus. Here we affirm this prediction, showing that the C termini of epitope-tagged, full-length human, rabbit, and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane. Truncation of these proteins at or near the predicted junction between the 13th TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane and (ii) interacted with antibodies only after permeabilization of the plasma membrane. CHO cells expressing rbMate1 truncated at residue Gly-545 supported levels of pH-sensitive transport similar to that of cells expressing the full-length protein. Although the high transport rate of the Gly-545 truncation mutant was associated with higher levels of membrane expression (than full-length MATE1), suggesting the 13th TMH may influence substrate translocation, the selectivity profile of the mutant indicated that TMH13 has little impact on ligand binding. We conclude that the functional core of MATE1 consists of 12 (not 13) TMHs. Therefore, we used the x-ray structure of NorM to develop a homology model of the first 12 TMHs of MATE1. The model proved to be stable in molecular dynamic simulations and agreed with topology evident from preliminary cysteine scanning of intracellular versus extracellular loops. PMID:22722930

Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Němcová-Fürstová, Vlasta, E-mail: vlasta.furstova@

Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100 nM and 300 nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublinesmore » of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells. - Highlights: • Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed. • SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes. • Some taxanes are able to overcome developed resistance to paclitaxel. • Paclitaxel resistance was associated with increased levels of ABCB1 and ABCC3 protein. • ABCB1 silencing increased significantly sensitivity to both paclitaxel and doxorubicin.« less

Pathobiological implications of the d16HER2 splice variant for stemness and aggressiveness of HER2-positive breast cancer

PubMed Central

Castagnoli, L; Ghedini, G C; Koschorke, A; Triulzi, T; Dugo, M; Gasparini, P; Casalini, P; Palladini, A; Iezzi, M; Lamolinara, A; Lollini, P L; Nanni, P; Chiodoni, C; Tagliabue, E; Pupa, S M

2017-01-01

We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial–mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene. PMID:27641338

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

PubMed Central

Curriu, Marta; Fausther-Bovendo, Hughes; Pernas, María; Massanella, Marta; Carrillo, Jorge; Cabrera, Cecilia; López-Galíndez, Cecilio; Clotet, Bonaventura; Debré, Patrice; Vieillard, Vincent; Blanco, Julià

2012-01-01

Background CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. Methods We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. Results Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. Conclusions Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41. PMID:22312424

Structure and expression profiling of a novel calcium-dependent protein kinase gene, CDPK3a, in leaves, stems, grapes, and cell cultures of wild-growing grapevine Vitis amurensis Rupr.

PubMed

Kiselev, K V; Dubrovina, A S; Shumakova, O A; Karetin, Y A; Manyakhin, A Y

2013-03-01

KEY MESSAGE : VaCDPK3a is actively expressed in leaves, stems, inflorescences, and berries of Vitis amurensis and may act as a positive growth regulator, but is not involved in the regulation of resveratrol biosynthesis. Calcium-dependent protein kinases (CDPKs) are known to play important roles in plant development and defense against biotic and abiotic stresses. It has previously been shown that CDPK3a is the predominant CDPK transcript in cell cultures of wild-growing grapevine Vitis amurensis Rupr., which is known to possess high resistance against environmental stresses and to produce resveratrol, a polyphenol with valuable pharmacological effects. In this study, we aimed to define the full cDNA sequence of VaCDPK3a and analyze its organ-specific expression, responses to plant hormones, temperature stress and exogenous NaCl, and the effects of VaCDPK3a overexpression on biomass accumulation and resveratrol content in V. amurensis calli. VaCDPK3a was actively expressed in all analyzed V. amurensis organs and tissues and was not transcriptionally regulated by salt and temperature stresses. The highest VaCDPK3a expression was detected in young leaves and the lowest in stems. A reduction in the VaCDPK3a expression correlated with a lower rate of biomass accumulation and higher resveratrol content in calli of V. amurensis under different growth conditions. Overexpression of the VaCDPK3a gene in the V. amurensis calli significantly increased cell growth for a short period of time but did not have an effect on resveratrol production. Further subculturing of the transformed calli resulted in cell death and a decrease in expression of the endogenous VaCDPK3a. The data suggest that while VaCDPK3a acts as a positive regulator of V. amurensis cell growth, it is not involved in the signaling pathway regulating resveratrol biosynthesis and resistance to salt and temperature stresses.

L1 stimulation of human glioma cell motility correlates with FAK activation

PubMed Central

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A.; Boulos, Magdy I.; Kappes, John C.

2011-01-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes. PMID:21373966

L1 stimulation of human glioma cell motility correlates with FAK activation.

PubMed

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A; Boulos, Magdy I; Kappes, John C; Galileo, Deni S

2011-10-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

Mycobacterium tuberculosis GroEL2 Modulates Dendritic Cell Responses.

PubMed

Georgieva, Maria; Sia, Jonathan Kevin; Bizzell, Erica; Madan-Lala, Ranjna; Rengarajan, Jyothi

2018-02-01

Mycobacterium tuberculosis successfully subverts the host immune response to promote disease progression. In addition to its known intracellular niche in macrophages, M. tuberculosis interferes with the functions of dendritic cells (DCs), which are the primary antigen-presenting cells of the immune system. We previously showed that M. tuberculosis dampens proinflammatory responses and impairs DC functions through the cell envelope-associated serine protease Hip1. Here we present data showing that M. tuberculosis GroEL2, a substrate of Hip1, modulates DC functions. The full-length GroEL2 protein elicited robust proinflammatory responses from DCs and promoted DC maturation and antigen presentation to T cells. In contrast, the cleaved form of GroEL2, which predominates in M. tuberculosis , was poorly immunostimulatory and was unable to promote DC maturation and antigen presentation. Moreover, DCs exposed to full-length, but not cleaved, GroEL2 induced strong antigen-specific gamma interferon (IFN-γ), interleukin-2 (IL-2), and IL-17A cytokine responses from CD4 + T cells. Moreover, the expression of cleaved GroEL2 in the hip1 mutant restored the robust T cell responses to wild-type levels, suggesting that proteolytic cleavage of GroEL2 allows M. tuberculosis to prevent optimal DC-T cell cross talk during M. tuberculosis infection. Copyright © 2018 American Society for Microbiology.

Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

PubMed

Nik-Ahd, Farnoosh; Bertoni, Carmen

2014-07-01

Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients. © 2014 AlphaMed Press.

MicroRNA-200c-141 and ∆Np63 are required for breast epithelial differentiation and branching morphogenesis.

PubMed

Hilmarsdóttir, Bylgja; Briem, Eirikur; Sigurdsson, Valgardur; Franzdóttir, Sigrídur Rut; Ringnér, Markus; Arason, Ari Jon; Bergthorsson, Jon Thor; Magnusson, Magnus Karl; Gudjonsson, Thorarinn

2015-07-15

The epithelial compartment of the breast contains two lineages, the luminal- and the myoepithelial cells. D492 is a breast epithelial cell line with stem cell properties that forms branching epithelial structures in 3D culture with both luminal- and myoepithelial differentiation. We have recently shown that D492 undergo epithelial to mesenchymal transition (EMT) when co-cultured with endothelial cells. This 3D co-culture model allows critical analysis of breast epithelial lineage development and EMT. In this study, we compared the microRNA (miR) expression profiles for D492 and its mesenchymal-derivative D492M. Suppression of the miR-200 family in D492M was among the most profound changes observed. Exogenous expression of miR-200c-141 in D492M reversed the EMT phenotype resulting in gain of luminal but not myoepithelial differentiation. In contrast, forced expression of ∆Np63 in D492M restored the myoepithelial phenotype only. Co-expression of miR-200c-141 and ∆Np63 in D492M restored the branching morphogenesis in 3D culture underlining the requirement for both luminal and myoepithelial elements for obtaining full branching morphogenesis in breast epithelium. Introduction of a miR-200c-141 construct in both D492 and D492M resulted in resistance to endothelial induced EMT. In conclusion, our data suggests that expression of miR-200c-141 and ∆Np63 in D492M can reverse EMT resulting in luminal- and myoepithelial differentiation, respectively, demonstrating the importance of these molecules in epithelial integrity in the human breast. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Use of anoctamin 1 (ANO1) to evaluate interstitial cells of Cajal in Hirschsprung's disease.

PubMed

Coyle, David; Kelly, Danielle A M; O'Donnell, Anne Marie; Gillick, John; Puri, Prem

2016-02-01

Interstitial cells of Cajal (ICCs) are pacemaker cells involved in facilitating neurotransmission and the generation of slow electrical waves necessary for colonic peristalsis. Their distribution has been found to be abnormal in the aganglionic and ganglionic colon in Hirschsprung's disease (HSCR) using c-kit-labelling. Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel thought to be specifically expressed on ICCs. Unlike c-kit, it plays a key role in ICC pacemaker activity. We aimed to investigate the utility of ANO1 in evaluating the colonic ICC network in HSCR. We collected full-length pull-through specimens from children with HSCR (n = 10). Control colon specimens were collected at colostomy closure in children with anorectal malformation (n = 6). The distribution of ANO1 and c-kit expression was evaluated using immunofluorescence and confocal microscopy. ANO1 expression was quantified using Western blot analysis. ANO1 was not expressed on 23 % of c-kit immuno-positive cells in the circular muscle; however, 100 % of ANO1-positive ICCs were c-kit positive. The distribution of ANO1-positive ICCs was sparse in aganglionic colon, with a modest reduction in ICCs seen in the ganglionic colon in HSCR compared to controls (p = 0.044). ANO1 protein expression was reduced in aganglionic colon but similar in ganglionic colon relative to controls. ANO1 is preferential to c-kit in evaluating the ICC network in HSCR due to its specificity and functional importance. Abnormal distribution of ANO1-positive ICCs in the ganglionic colon in HSCR may contribute to persistent bowel symptoms in some patients after pull-through surgery.

Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium.

PubMed

Moffatt, Pierre; Smith, Charles E; St-Arnaud, René; Simmons, Darrin; Wright, J Timothy; Nanci, Antonio

2006-10-01

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

Small and big Hodgkin-Reed-Sternberg cells of Hodgkin lymphoma cell lines L-428 and L-1236 lack consistent differences in gene expression profiles and are capable to reconstitute each other.

PubMed

Rengstl, Benjamin; Kim, Sooji; Döring, Claudia; Weiser, Christian; Bein, Julia; Bankov, Katrin; Herling, Marco; Newrzela, Sebastian; Hansmann, Martin-Leo; Hartmann, Sylvia

2017-01-01

The hallmark of classical Hodgkin lymphoma (cHL) is the presence of giant, mostly multinucleated Hodgkin-Reed-Sternberg (HRS) cells. Whereas it has recently been shown that giant HRS cells evolve from small Hodgkin cells by incomplete cytokinesis and re-fusion of tethered sister cells, it remains unsolved why this phenomenon particularly takes place in this lymphoma and what the differences between these cell types of variable sizes are. The aim of the present study was to characterize microdissected small and giant HRS cells by gene expression profiling and to assess differences of clonal growth behavior as well as susceptibility toward cytotoxic intervention between these different cell types to provide more insight into their distinct cellular potential. Applying stringent filter criteria, only two differentially expressed genes between small and giant HRS cells, SHFM1 and LDHB, were identified. With looser filter criteria, 13 genes were identified to be differentially overexpressed in small compared to giant HRS cells. These were mainly related to energy metabolism and protein synthesis, further suggesting that small Hodgkin cells resemble the proliferative compartment of cHL. SHFM1, which is known to be involved in the generation of giant cells, was downregulated in giant RS cells at the RNA level. However, reduced mRNA levels of SHFM1, LDHB and HSPA8 did not translate into decreased protein levels in giant HRS cells. In cell culture experiments it was observed that the fraction of small and big HRS cells was adjusted to the basic level several days after enrichment of these populations via cell sorting, indicating that small and big HRS cells can reconstitute the full spectrum of cells usually observed in the culture. However, assessment of clonal growth of HRS cells indicated a significantly reduced potential of big HRS cells to form single cell colonies. Taken together, our findings pinpoint to strong similarities but also some differences between small and big HRS cells.

Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chu, Xiufeng; Zhang, Ting; Wang, Jie

2014-04-25

Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β havemore » been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers.« less

The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

PubMed Central

Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

2009-01-01

Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

Characterization of the gene encoding pinin/DRS/memA and evidence for its potential tumor suppressor function.

PubMed

Shi, Y; Ouyang, P; Sugrue, S P

2000-01-13

Several cell adhesion-related proteins have been shown to act as tumor-suppressors (TS) in the neoplastic progression of epithelial-derived tumors. Pinin/DRS/memA was first identified in our laboratory and it was shown to be a cell adhesion-related molecule. Our previous study demonstrated that restoration of pinin expression in transformed cells not only positively influenced cellular adhesive properties but also reversed the transformed phenotype to more epithelial-like. Here, we show by FISH analysis that the gene locus for pinin is within 14q13. The alignment of the pinin gene with STS markers localized the gene to the previously identified TS locus D14S75-D14S288. Northern analyses revealed diminished pinin mRNA in renal cell carcinomas (RCC) and certain cancer cell lines. Immunohistochemical examination of tumor samples demonstrated absent or greatly reduced pinin in transitional cell carcinoma (TCC) and RCC tumors. TCC-derived J82 cells as well as EcR-293 cells transfected with full-length pinin cDNA demonstrated inhibition of anchorage-independent growth of cells in soft agar. Furthermore, methylation analyses revealed that aberrant methylation of pinin CpG islands was correlated with decreased/absent pinin expression in a subset of tumor tissues. These data lend significant support to the hypothesis that pinin/DRS/memA may act as a tumor suppressor in certain types of cancers.

[Screening and identification of anoikis-resistant gene UBCH7 in esophageal cancer cells].

PubMed

Yang, Yang; Wang, Bo-Shi; Wang, Xiao-Min; Zhang, Yu; Wang, Ming-Rong; Jia, Xue-Mei

2012-02-01

Anoikis is a kind of programmed cell death induced by loss of extracellular matrix (ECM) adhesion, which is one of key factors for homestasis. Resistance to anoikis is required for tumor cell metastasis. We have previously shown several anoikis-resistance genes in esophageal squamous cell carcinoma (ESCC). In order to find novel anoikis-resistant genes in ESCC, we constructed retroviral cDNA library using total RNA from ESCC cell lines. NIH 3T3 cells, which are sensitive to anoikis, were infected with the library constructed. The cells were cultured in soft agar, and the clones which can survive in detached states were selected. The cDNAs inserted into the anoikis-resistant NIH3T3 clones were amplified using retroviral specific primers. Sequencing analysis showed that a cDNA fragment inserted into the anoikis-resistant clone contains full coding sequence (ORF) of human UBCH7/UBE2L3 gene. By infection with retrovirus encoding UBCH7 ORF (pMSCV-UBCH7), forced expression of UBCH7 increased the anoikis-resistance of NIH3T3 cells. More importantly, knockdown of UBCH7 expression by siRNA transfection reduced the anoikis-resistant ability of esophageal cancer MLuC1 cells. The data suggest that UBCH7/UBE2L3 gene would be involved in anoikis-resistance in ESCC.

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

PubMed

Nhan, Nguyen Thanh; Gonzalez de Valdivia, Ernesto; Gustavsson, Martin; Hai, Truong Nam; Larsson, Gen

2011-04-11

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).

PubMed

Schroer, Adam B; Gross, Joshua D; Kaski, Shane W; Wix, Kim; Siderovski, David P; Vandenbeuch, Aurelie; Setola, Vincent

2018-05-23

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

PubMed

Sessa, F; Bonato, M; Frigerio, B; Capella, C; Solcia, E; Prat, M; Bara, J; Samloff, I M

1990-06-01

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

PubMed

Zhang, Yuyao; Ma, Xiuli; Huang, Bing; Li, Yufeng; Yu, Kexiang; Li, Jianliang; Liu, Cunxia; Han, Hongyu; Cui, Yanshun

2015-04-04

To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Bmi-1-targeting suppresses osteosarcoma aggressiveness through the NF-κB signaling pathway

PubMed Central

Liu, Jiaguo; Luo, Bin; Zhao, Meng

2017-01-01

Bone cancer is one of the most lethal malignancies and the specific causes of tumor initiation are not well understood. B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been reported to be associated with the initiation and progression of osteosarcoma, and as a prognostic indicator in the clinic. In the current study, a full-length antibody targeting Bmi-1 (AbBmi-1) was produced and the preclinical value of Bmi-1-targeted therapy was evaluated in bone carcinoma cells and tumor xenograft mice. The results indicated that the Bmi-1 expression level was markedly upregulated in bone cancer cell lines, and inhibition of Bmi-1 by AbBmi-1 reduced the invasiveness and migration of osteosarcoma cells. Overexpression of Bmi-1 promoted proliferation and angiogenesis, and increased apoptosis resistance induced by cisplatin via the nuclear factor-κB (NF-κB) signal pathway. In addition, AbBmi-1 treatment inhibited the tumorigenicity of osteosarcoma cells in vivo. Furthermore, AbBmi-1 blocked NF-κB signaling and reduced MMP-9 expression. Furthermore, Bmi-1 promoted osteosarcoma tumor growth, whereas AbBmi-1 significantly inhibited osteosarcoma tumor growth in vitro and in vivo. Notably, AbBmi-1 decreased the percentages of Ki67-positive cells and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in tumors compared with Bmi-1-treated and PBS controls. Notably, MMP-9 and NF-κB expression were downregulated by treatment with AbBmi-1 in MG-63 osteosarcoma cells. In conclusion, the data provides evidence that AbBmi-1 inhibited the progression of osteosarcoma, suggesting that AbBmi-1 may be a novel anti-cancer agent through the inhibition of Bmi-1 via activating the NF-κB pathway in osteosarcoma. PMID:28983587

Distinct pathways leading to TDP-43-induced cellular dysfunctions.

PubMed

Yamashita, Makiko; Nonaka, Takashi; Hirai, Shinobu; Miwa, Akiko; Okado, Haruo; Arai, Tetsuaki; Hosokawa, Masato; Akiyama, Haruhiko; Hasegawa, Masato

2014-08-15

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component protein of inclusions found in brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the molecular mechanisms by which TDP-43 causes neuronal dysfunction and death remain unknown. Here, we report distinct cytotoxic effects of full-length TDP-43 (FL-TDP) and its C-terminal fragment (CTF) in SH-SY5Y cells. When FL-TDP was overexpressed in the cells using a lentiviral system, exogenous TDP-43, like endogenous TDP-43, was expressed mainly in nuclei of cells without any intracellular inclusions. However, these cells showed striking cell death, caspase activation and growth arrest at G2/M phase, indicating that even simple overexpression of TDP-43 induces cellular dysfunctions leading to apoptosis. On the other hand, cells expressing TDP-43 CTF showed cytoplasmic aggregates but without significant cell death, compared with cells expressing FL-TDP. Confocal microscopic analyses revealed that RNA polymerase II (RNA pol II) and several transcription factors, such as specificity protein 1 and cAMP-response-element-binding protein, were co-localized with the aggregates of TDP-43 CTF, suggesting that sequestration of these factors into TDP-43 aggregates caused transcriptional dysregulation. Indeed, accumulation of RNA pol II at TDP-43 inclusions was detected in brains of patients with FTLD-TDP. Furthermore, apoptosis was not observed in affected neurons of FTLD-TDP brains containing phosphorylated and aggregated TDP-43 pathology. Our results suggest that different pathways of TDP-43-induced cellular dysfunction may contribute to the degeneration cascades involved in the onset of ALS and FTLD-TDP. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway.

PubMed

Matsuzawa, M; Sheu, T-J; Lee, Y-J; Chen, M; Li, T-F; Huang, C T; Holz, J D; Puzas, J E

2009-06-01

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

MINA controls proliferation and tumorigenesis of glioblastoma by epigenetically regulating cyclins and CDKs via H3K9me3 demethylation.

PubMed

Huang, M-Y; Xuan, F; Liu, W; Cui, H-J

2017-01-19

It is generally known that histone demethylases regulate gene transcription by altering the methylate status on histones, but their roles in cancers and the underlying molecular mechanisms still remain unclear. MYC-induced nuclear antigen (MINA) is reported to be a histone demethylase and highly expressed in many cancers. Here, for the first time, we show that MINA is involved in glioblastoma carcinogenesis and reveal the probable mechanisms of it in cell-cycle control. Kaplan-Meier analysis of progression-free survival showed that high MINA expression was strongly correlated with poor outcome and advancing tumor stage. MINA knockdown significantly repressed the cell proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo that were rescued by overexpressing the full-length MINA afterwards. Microarray analysis after knockdown of MINA revealed that MINA probably regulated glioblastoma carcinogenesis through the predominant cell-cycle pathways. Further investigation showed that MINA deficiency led to a cell-cycle arrest in G1 and G2 phases. And among the downstream genes, we found that cyclins and cyclin-dependent kinases were directly activated by MINA via the demethylation of H3K9me3.

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3.

PubMed

Zhou, Yingying; Kang, Xilong; Xiong, Dan; Zhu, Shanshan; Zheng, Huijuan; Xu, Ying; Guo, Yaxin; Pan, Zhiming; Jiao, Xinan

2017-04-01

Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

PubMed

Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

2017-01-01

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Function of translationally controlled tumor protein (TCTP) in Eudrilus eugeniae regeneration

PubMed Central

Subramanian, Elaiya Raja; Gopi Daisy, Nino; Sudalaimani, Dinesh Kumar; Ramamoorthy, Kalidas; Balakrishnan, Subburathinam; Selvan Christyraj, Jackson Durairaj; Arumugaswami, Vaithilingaraja

2017-01-01

TCTP (Translationally Controlled Tumour Protein) is a multifunctional protein that plays a role in the development, immune system, tumour reversion, and maintenance of stem cells. The mRNA of the Tpt1 gene is over-expressed during liver regeneration. But, the function of the protein in regeneration is not known. To study the role of the protein in regeneration, the earthworm Eudrilus eugeniae was chosen. First, the full length cDNA of the Tpt1 gene was sequenced. The size of the cDNA is 504 bp and the protein has 167 amino acids. The highest level of TCTP expression was documented in the worm after three days of regeneration. The protein was found to be expressed specifically in the epithelial layer of the skin. During regeneration, the protein expression was found to be the highest at the tip of blastema. The pharmacological suppression of TCTP using nutlin-3 and TCTP RNAi experiments resulted in the failure of the regeneration process. The suppression of TCTP caused the arrest of proliferation in posterior amputated worms. The severe cell death was documented in the amputated region of nutlin-3 injected worm. The silencing of TCTP has blocked the modification of clitellar segments. The experiments confirm that TCTP has major functions in the upstream signalling of cell proliferation in the early regeneration process in E. eugeniae. PMID:28403226

Function of translationally controlled tumor protein (TCTP) in Eudrilus eugeniae regeneration.

PubMed

Subramanian, Elaiya Raja; Gopi Daisy, Nino; Sudalaimani, Dinesh Kumar; Ramamoorthy, Kalidas; Balakrishnan, Subburathinam; Selvan Christyraj, Jackson Durairaj; Arumugaswami, Vaithilingaraja; Sivasubramaniam, Sudhakar

2017-01-01

TCTP (Translationally Controlled Tumour Protein) is a multifunctional protein that plays a role in the development, immune system, tumour reversion, and maintenance of stem cells. The mRNA of the Tpt1 gene is over-expressed during liver regeneration. But, the function of the protein in regeneration is not known. To study the role of the protein in regeneration, the earthworm Eudrilus eugeniae was chosen. First, the full length cDNA of the Tpt1 gene was sequenced. The size of the cDNA is 504 bp and the protein has 167 amino acids. The highest level of TCTP expression was documented in the worm after three days of regeneration. The protein was found to be expressed specifically in the epithelial layer of the skin. During regeneration, the protein expression was found to be the highest at the tip of blastema. The pharmacological suppression of TCTP using nutlin-3 and TCTP RNAi experiments resulted in the failure of the regeneration process. The suppression of TCTP caused the arrest of proliferation in posterior amputated worms. The severe cell death was documented in the amputated region of nutlin-3 injected worm. The silencing of TCTP has blocked the modification of clitellar segments. The experiments confirm that TCTP has major functions in the upstream signalling of cell proliferation in the early regeneration process in E. eugeniae.

Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

PubMed Central

Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

2016-01-01

P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strauss, W.M.; Dausman, J.; Beard, C.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protectionmore » analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.« less

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

PubMed

Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

2015-09-08

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Differentiation and Characterization of Myeloid Cells

PubMed Central

Gupta, Dipti; Shah, Hetavi Parag; Malu, Krishnakumar; Berliner, Nancy; Gaines, Peter

2015-01-01

Recent molecular studies of myeloid differentiation have utilized several in vitro models of myelopoiesis, generated from either ex vivo differentiated bone marrow progenitors or induced immortalized myeloid cell lines. Ex vivo differentiation begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca-1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid–induced myeloid development, however, both cell lines exhibit defects in the upregulation of late-expressed neutrophil-specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation, EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid, and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradial in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR. Together, these assays provide a solid foundation for in vitro investigations of myeloid development with either human or mouse models. PMID:24510620

Signal transduction in T lymphocytes in microgravity

NASA Technical Reports Server (NTRS)

Cogoli, A.

1997-01-01

More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma

PubMed Central

Siurala, Mikko; Havunen, Riikka; Saha, Dipongkor; Lumen, Dave; Airaksinen, Anu J.; Tähtinen, Siri; Cervera-Carrascon, Víctor; Bramante, Simona; Parviainen, Suvi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

2016-01-01

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of 111In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies. PMID:27357626

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Four Novel Cellulose Synthase (CESA) Genes from Birch (Betula platyphylla Suk.) Involved in Primary and Secondary Cell Wall Biosynthesis

PubMed Central

Liu, Xuemei; Wang, Qiuyu; Chen, Pengfei; Song, Funan; Guan, Minxiao; Jin, Lihua; Wang, Yucheng; Yang, Chuanping

2012-01-01

Cellulose synthase (CESA), which is an essential catalyst for the generation of plant cell wall biomass, is mainly encoded by the CesA gene family that contains ten or more members. In this study; four full-length cDNAs encoding CESA were isolated from Betula platyphylla Suk., which is an important timber species, using RT-PCR combined with the RACE method and were named as BplCesA3, −4, −7 and −8. These deduced CESAs contained the same typical domains and regions as their Arabidopsis homologs. The cDNA lengths differed among these four genes, as did the locations of the various protein domains inferred from the deduced amino acid sequences, which shared amino acid sequence identities ranging from only 63.8% to 70.5%. Real-time RT-PCR showed that all four BplCesAs were expressed at different levels in diverse tissues. Results indicated that BplCESA8 might be involved in secondary cell wall biosynthesis and floral development. BplCESA3 appeared in a unique expression pattern and was possibly involved in primary cell wall biosynthesis and seed development; it might also be related to the homogalacturonan synthesis. BplCESA7 and BplCESA4 may be related to the formation of a cellulose synthase complex and participate mainly in secondary cell wall biosynthesis. The extremely low expression abundance of the four BplCESAs in mature pollen suggested very little involvement of them in mature pollen formation in Betula. The distinct expression pattern of the four BplCesAs suggested they might participate in developments of various tissues and that they are possibly controlled by distinct mechanisms in Betula. PMID:23202892

Virus-specific CD8+ T cells infiltrate melanoma lesions and retain function independent of PD-1 expression

PubMed Central

Erkes, Dan A.; Smith, Corinne J.; Wilski, Nicole A.; Caldeira-Dantas, Sofia; Mohgbeli, Toktam; Snyder, Christopher M.

2017-01-01

It is well known that CD8+ tumor infiltrating lymphocytes (TIL) are correlated with positive prognoses in cancer patients and used to determine efficacy of immune therapies. While it is generally assumed that CD8+ TIL will be tumor associated antigen (TAA)-specific, it is unknown whether CD8+ T cells with specificity for common pathogens also infiltrate tumors. If so, the presence of these T cells could alter the interpretation of prognostic and diagnostic TIL assays. We compared TAA-specific and virus-specific CD8+ T cells in the same tumors using murine cytomegalovirus (MCMV), a herpesvirus that causes a persistent/latent infection, and Vaccinia virus (VacV), a poxvirus that is cleared by the host. Virus-specific CD8+ TIL migrated into cutaneous melanoma lesions during acute infection with either virus, as well as after a cleared VacV infection, and during a persistent/latent MCMV infection. Virus-specific TILs developed independent of viral antigen in the tumor and interestingly, expressed low or intermediate levels of full-length PD-1 in the tumor environment. Importantly, PD-1 expression could be markedly induced by antigen, but did not correlate with dysfunction for virus-specific TIL, in sharp contrast to TAA-specific TIL in the same tumors. These data suggest that CD8+ TIL can reflect an individual's immune status, rather than exclusively representing TAA-specific T cells, and that PD-1 expression on CD8+ TIL is not always associated with repeated antigen encounter or dysfunction. Thus, functional virus-specific CD8+ TIL could skew the results of prognostic or diagnostic TIL assays. PMID:28202614

Spatiotemporal relationship of embryonic cholinesterases with cell proliferation in chicken brain and eye.

PubMed Central

Layer, P G; Sporns, O

1987-01-01

Close relationships between acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, true cholinesterase, EC, 3.1.1.7) and butyrylcholinesterase (BtChoEase, acylcholine acylhydrolase, pseudocholinesterase, EC, 3.1.1.8) with cell proliferation were observed in the early chicken brain. These include the following: BtChoEase is transiently accumulating in patchy fashion on the ventricular side of the neuroepithelium shortly before AcChoEase appears in cell bodies along the opposing mantle layer. The amount of BtChoEase in retina and brain is greatest in the early phase (E3-E5, or incubation periods of 3-5 days); in retina it decreases about 2 days later than in brain. However, AcChoEase expression increases with time, in inverse order to that of BtChoEase. In both tissues decrease of cell proliferation is closely followed by decrease in BtChoEase. A double-labeling technique of cholinesterase staining together with [3H]thymidine autoradiography reveals proliferation zones that are diffusely stained by BtChoEase but not by AcChoEase. Patches intensely stained for BtChoEase accompany clusters of cells in final stages of mitosis on their way to the differentiation zone, where they begin expressing AcChoEase. By applying different thymidine pulses, we identify an 11-hr lag from the last thymidine-uptake to full AcChoEase expression. (iv) These findings are confirmed by studying lens development, where areas of proliferation and differentiation are well separated. The spatiotemporal pattern of the transition of neuroblasts from a proliferating into a differentiating state correlates with the expression of BtChoEase just before and during mitosis and that of AcChoEase about 11 hr after mitosis. Thus cholinesterases could be involved in the regulation of this transition. Images PMID:3467355