Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

PubMed

Granja, Sara; Marchiq, Ibtissam; Le Floch, Renaud; Moura, Conceição Souto; Baltazar, Fátima; Pouysségur, Jacques

2015-03-30

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

Cardiomyocyte-specific desmin rescue of desmin null cardiomyopathy excludes vascular involvement.

PubMed

Weisleder, Noah; Soumaka, Elisavet; Abbasi, Shahrzad; Taegtmeyer, Heinrich; Capetanaki, Yassemi

2004-01-01

Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.

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

Simoes, Maria L.; Hockley, Sarah L.; Schwerdtle, Tanja

Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy. These tumours contain TP53 mutations and over-express TP53. We compared transcriptional and translational responses of two isogenic HCT116 cell lines, one expressing TP53 (p53-WT) and the other with this gene knocked out (p53-null), to treatment with aristolochic acid I (AAI) (50-100 {mu}M) for 6-48 h. Modulation of 118 genes was observed in p53-WT cells and 123 genes in p53-null cells. Some genes, including INSIG1, EGR1, CAV1, LCN2 and CCNG1, were differentially expressed in the two cell lines. CDKN1A was selectively up-regulated in p53-WT cells, leadingmore » to accumulation of TP53 and CDKN1A. Apoptotic signalling, measured by caspase-3 and -7 activity, was TP53-dependent. Both cell types accumulated in S phase, suggesting that AAI-DNA adducts interfere with DNA replication, independently of TP53 status. The oncogene MYC, frequently over-expressed in urothelial tumours, was up-regulated by AAI, whereas FOS was down-regulated. Observed modulation of genes involved in endocytosis, e.g. RAB5A, may be relevant to the known inhibition of receptor-mediated endocytosis, an early sign of AA-mediated proximal tubule injury. AAI-DNA adduct formation was significantly greater in p53-WT cells than in p53-null cells. Collectively, phenotypic anchoring of the AAI-induced expression profiles to DNA adduct formation, cell-cycle parameters, TP53 expression and apoptosis identified several genes linked to these biological outcomes, some of which are TP53-dependent. These results strengthen the importance of TP53 in AA-induced cancer, and indicate that other alterations, e.g. to MYC oncogenic pathways, may also contribute.« less

Influence of ribosomal protein L39-L in the drug resistance mechanisms of lacrimal gland adenoid cystic carcinoma cells.

PubMed

Ye, Qing; Ding, Shao-Feng; Wang, Zhi-An; Feng, Jie; Tan, Wen-Bin

2014-01-01

Cancer constitutes a key pressure on public health regardless of the economy state in different countries. As a kind of highly malignant epithelial tumor, lacrimal gland adenoid cystic carcinoma can occur in any part of the body, such as salivary gland, submandibular gland, trachea, lung, breast, skin and lacrimal gland. Chemotherapy is one of the key treatment techniques, but drug resistance, especially MDR, seriously blunts its effects. As an element of the 60S large ribosomal subunit, the ribosomal protein L39-L gene appears to be documented specifically in the human testis and many human cancer samples of different origins. Total RNA of cultured drug-resistant and susceptible lacrimal gland adenoid cystic carcinoma cells was seperated, and real time quantitative RT-PCR were used to reveal transcription differences between amycin resistant and susceptible strains of lacrimal gland adenoid cystic carcinoma cells. Viability assays were used to present the amycin resistance difference in a RPL39-L transfected lacrimal gland adenoid cystic carcinoma cell line as compared to control vector and null-transfected lacrimal gland adenoid cystic carcinoma cell lines. The ribosomal protein L39-L transcription level was 6.5-fold higher in the drug-resistant human lacrimal gland adenoid cystic carcinoma cell line than in the susceptible cell line by quantitative RT-PCR analysis. The ribosomal protein L39-L transfected cells revealed enhanced drug resistance compared to plasmid vector-transfected or null-transfected cells as determined by methyl tritiated thymidine (3H-TdR) incorporation. The ribosomal protein L39-L gene could possibly have influence on the drug resistance mechanism of lacrimal gland adenoid cystic carcinoma cells.

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

The appearance, motion, and disappearance of three-dimensional magnetic null points

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

Murphy, Nicholas A., E-mail: namurphy@cfa.harvard.edu; Parnell, Clare E.; Haynes, Andrew L.

2015-10-15

While theoretical models and simulations of magnetic reconnection often assume symmetry such that the magnetic null point when present is co-located with a flow stagnation point, the introduction of asymmetry typically leads to non-ideal flows across the null point. To understand this behavior, we present exact expressions for the motion of three-dimensional linear null points. The most general expression shows that linear null points move in the direction along which the magnetic field and its time derivative are antiparallel. Null point motion in resistive magnetohydrodynamics results from advection by the bulk plasma flow and resistive diffusion of the magnetic field,more » which allows non-ideal flows across topological boundaries. Null point motion is described intrinsically by parameters evaluated locally; however, global dynamics help set the local conditions at the null point. During a bifurcation of a degenerate null point into a null-null pair or the reverse, the instantaneous velocity of separation or convergence of the null-null pair will typically be infinite along the null space of the Jacobian matrix of the magnetic field, but with finite components in the directions orthogonal to the null space. Not all bifurcating null-null pairs are connected by a separator. Furthermore, except under special circumstances, there will not exist a straight line separator connecting a bifurcating null-null pair. The motion of separators cannot be described using solely local parameters because the identification of a particular field line as a separator may change as a result of non-ideal behavior elsewhere along the field line.« less

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status

PubMed Central

Floch, Renaud Le; Moura, Conceição Souto

2015-01-01

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function. To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via ‘Zinc-Finger Nucleases’. The three homozygous BSG−/− cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG−/− cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin. PMID:25894929

IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells

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

Li Benhui; Yang Xianzi; Department of Medical Oncology, Taihe Hospital, Yunyang Medical College, Shiyan, Hubei 442000

2008-05-02

IL-4-induced Stat6 signaling is active in a variety of cell types and plays a role in cell proliferation/growth and resistance to apoptosis. Using EMSA, we identified differential IL-4/Stat6 activities in colorectal cancer cell lines, HT-29 being active Stat6{sup high} phenotype and Caco-2 being defective Stat6{sup null} phenotype, respectively. Active Stat6{sup high} HT-29 cells exhibited resistance to apoptosis by flowcytometry and aggressive metastasis by Transwell assay compared with defective Stat6{sup null} Caco-2 cells. Comparing one another using RT-PCR, Stat6{sup high} HT-29 cells expressed more mRNA of anti-apoptotic and pro-metastatic genes Survivin, MDM2, and TMPRSS4, while Stat6{sup null} Caco-2 cells expressed moremore » mRNA of pro-apoptotic and anti-metastatic genes BAX, CAV1, and P53, respectively. This is the first study describing correlations of IL-4/Stat6 activities with apoptosis and metastasis in colon cancer. These findings, together with the observation of constitutive Stat6 activation in many human malignancies, suggest that Stat6 activities could be a biomarker for cancer cell's invasive/metastatic capability.« less

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.

PubMed

Wasielewski, Marijke; Hanifi-Moghaddam, Pejman; Hollestelle, Antoinette; Merajver, Sofia D; van den Ouweland, Ans; Klijn, Jan G M; Ethier, Stephen P; Schutte, Mieke

2009-01-01

The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.

Urea transporter UT-B deletion induces DNA damage and apoptosis in mouse bladder urothelium.

PubMed

Dong, Zixun; Ran, Jianhua; Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders.

Urea Transporter UT-B Deletion Induces DNA Damage and Apoptosis in Mouse Bladder Urothelium

PubMed Central

Zhou, Hong; Chen, Jihui; Lei, Tianluo; Wang, Weiling; Sun, Yi; Lin, Guiting; Bankir, Lise; Yang, Baoxue

2013-01-01

Background Previous studies found that urea transporter UT-B is abundantly expressed in bladder urothelium. However, the dynamic role of UT-B in bladder urothelial cells remains unclear. The objective of this study is to evaluate the physiological roles of UT-B in bladder urothelium using UT-B knockout mouse model and T24 cell line. Methodology/Principal Findings Urea and NO measurement, mRNA expression micro-array analysis, light and transmission electron microscopy, apoptosis assays, DNA damage and repair determination, and intracellular signaling examination were performed in UT-B null bladders vs wild-type bladders and in vitro T24 epithelial cells. UT-B was highly expressed in mouse bladder urothelium. The genes, Dcaf11, MCM2-4, Uch-L1, Bnip3 and 45 S pre rRNA, related to DNA damage and apoptosis were significantly regulated in UT-B null urothelium. DNA damage and apoptosis highly occurred in UT-B null urothelium. Urea and NO levels were significantly higher in UT-B null urothelium than that in wild-type, which may affect L-arginine metabolism and the intracellular signals related to DNA damage and apoptosis. These findings were consistent with the in vitro study in T24 cells that, after urea loading, exhibited cell cycle delay and apoptosis. Conclusions/Significance UT-B may play an important role in protecting bladder urothelium by balancing intracellular urea concentration. Disruption of UT-B function induces DNA damage and apoptosis in bladder, which can result in bladder disorders. PMID:24204711

Expression of Basigin in Reproductive Tissues of Oestrogen Receptor-α or –β Null Mice

PubMed Central

Chen, Li; Bi, Jiajia; Nakai, Masaaki; Bunick, David; Couse, John F.; Korach, Kenneth S.; Nowak, Romana A.

2016-01-01

Basigin plays important roles in both male and female reproduction because basigin (Bsg) null male and female mice are infertile. The aim of the present study was to determine whether basigin expression in reproductive organs requires oestrogen receptor (ER) α or ERβ. Expression of basigin protein in the testis, ovary and male and female reproductive tracts was studied in adult wild type, ERα-null (αERKO) and ERβ-null (βERKO) mice by immunohistochemistry and immunoblotting. Basigin mRNA levels in ovary and uterus were examined by quantitative RT-PCR. In females, basigin protein expression was observed mainly in granulosa and interstitial cells of the ovary and epithelial cells of the proximal oviduct in all genotypes. Basigin protein was also expressed in the uterine epithelium at prooestrus and oestrus in WT and βERKO mice but not in αERKO mice. However, a higher level of basigin mRNA was observed in uteri of αERKO mice compared with WT and βERKO mice. In males, basigin was expressed in Leydig cells and all germ cells except spermatogonia in all genotypes. Basigin was present in epithelial cells lining the efferent ductules in WT and βERKO mice but expression was greatly reduced in αERKO mice. In epididymal ducts, basigin expression was observed in epithelial cells in the caput and cauda in all genotypes. These data suggest that expression of basigin protein requires ERα, but not ERβ, in the uterus and efferent ductules, but is independent of ER in the ovary, oviduct, testis and epididymis. PMID:20388736

ON THE NATURE OF RECONNECTION AT A SOLAR CORONAL NULL POINT ABOVE A SEPARATRIX DOME

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

Pontin, D. I.; Priest, E. R.; Galsgaard, K., E-mail: dpontin@maths.dundee.ac.uk

2013-09-10

Three-dimensional magnetic null points are ubiquitous in the solar corona and in any generic mixed-polarity magnetic field. We consider magnetic reconnection at an isolated coronal null point whose fan field lines form a dome structure. Using analytical and computational models, we demonstrate several features of spine-fan reconnection at such a null, including the fact that substantial magnetic flux transfer from one region of field line connectivity to another can occur. The flux transfer occurs across the current sheet that forms around the null point during spine-fan reconnection, and there is no separator present. Also, flipping of magnetic field lines takesmore » place in a manner similar to that observed in the quasi-separatrix layer or slip-running reconnection.« less

Dual mTORC1/2 inhibition induces anti-proliferative effect in NF1-associated plexiform neurofibroma and malignant peripheral nerve sheath tumor cells

PubMed Central

Hivelin, Mikael; Nusbaum, Patrick; Hubas, Arnaud; Laurendeau, Ingrid; Lantieri, Laurent; Wolkenstein, Pierre; Vidaud, Michel; Pasmant, Eric; Chapuis, Nicolas; Parfait, Béatrice

2016-01-01

Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines (90-8, 88-14 and 96-2), STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive “active-site” mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies. PMID:26840085

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38

PubMed Central

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-01-01

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug. PMID:25010984

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

PubMed

Weilbacher, A; Gutekunst, M; Oren, M; Aulitzky, W E; van der Kuip, H

2014-07-10

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Metformin attenuates ovarian cancer cell growth in an AMP-kinase dispensable manner

PubMed Central

Rattan, R; Giri, S; Hartmann, LC; Shridhar, V

2011-01-01

Abstract Metformin, the most widely used drug for type 2 diabetes activates 59 adenosine monophosphate (AMP)-activated protein kinase (AMPK), which regulates cellular energy metabolism. Here, we report that ovarian cell lines VOSE, A2780, CP70, C200, OV202, OVCAR3, SKOV3ip, PE01 and PE04 predominantly express -α1, -β1, -γ1 and -γ2 isoforms of AMPK subunits. Our studies show that metformin treatment (1) significantly inhibited proliferation of diverse chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3, SKVO3ip, PE01 and PE04), (2) caused cell cycle arrest accompanied by decreased cyclin D1 and increased p21 protein expression, (3) activated AMPK in various ovarian cancer cell lines as evident from increased phosphorylation of AMPKα and its downstream substrate; acetyl co-carboxylase (ACC) and enhanced β-oxidation of fatty acid and (4) attenuated mTOR-S6RP phosphorylation, inhibited protein translational and lipid biosynthetic pathways, thus implicating metformin as a growth inhibitor of ovarian cancer cells. We also show that metformin-mediated effect on AMPK is dependent on liver kinase B1 (LKB1) as it failed to activate AMPK-ACC pathway and cell cycle arrest in LKB1 null mouse embryo fibroblasts (mefs). This observation was further supported by using siRNA approach to down-regulate LKB1 in ovarian cancer cells. In contrast, met formin inhibited cell proliferation in both wild-type and AMPKα1/2 null mefs as well as in AMPK silenced ovarian cancer cells. Collectively, these results provide evidence on the role of metformin as an anti-proliferative therapeutic that can act through both AMPK-dependent as well as AMPK-independent pathways. PMID:19874425

BMP suppresses PTEN expression via RAS/ERK signaling.

PubMed

Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

Cyclin A2 promotes DNA repair in the brain during both development and aging.

PubMed

Gygli, Patrick E; Chang, Joshua C; Gokozan, Hamza N; Catacutan, Fay P; Schmidt, Theresa A; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J; Czeisler, Catherine; Otero, José J

2016-07-01

Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation inCCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.

Hybridization of downregulated-COMT transgenic switchgrass lines with field-selected switchgrass for improved biomass traits

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

Baxter, Holly L.; Alexander, Lisa W.; Mazarei, Mitra

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. For instance, downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In this present study we sought to further improve biomass characteristics by crossing the downregulated COMT T 1 lines with high-yielding switchgrass accessions in two genetic backgrounds ('Alamo' and 'Kanlow'). Crosses and T 2 progeny analyses were made under greenhouse conditions to assess maternal effects, plant morphology and yield, and cell wall traits. Female parent type influenced morphology, but had no effect on cell wall traits. Tmore » 2 hybrids produced with T 1 COMT-downregulated switchgrass as the female parent were taller, produced more tillers, and produced 63% more biomass compared with those produced using the field selected accession as the female parent. Transgene status (presence or absence of transgene) influenced both growth and cell wall traits. T 2 transgenic hybrids were 7% shorter 80 days after sowing and produced 43% less biomass than non-transgenic null-segregant hybrids. Cell wall-related differences included lower lignin content, reduced syringyl-to-guaiacyl (S/G) lignin monomer ratio, and a 12% increase in total sugar release in the T 2 transgenic hybrids compared to non-transgenic null segregants. This is the first study to evaluate the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits. Lastly, our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.« less

Hybridization of downregulated-COMT transgenic switchgrass lines with field-selected switchgrass for improved biomass traits

DOE PAGES

Baxter, Holly L.; Alexander, Lisa W.; Mazarei, Mitra; ...

2016-01-21

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. For instance, downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In this present study we sought to further improve biomass characteristics by crossing the downregulated COMT T 1 lines with high-yielding switchgrass accessions in two genetic backgrounds ('Alamo' and 'Kanlow'). Crosses and T 2 progeny analyses were made under greenhouse conditions to assess maternal effects, plant morphology and yield, and cell wall traits. Female parent type influenced morphology, but had no effect on cell wall traits. Tmore » 2 hybrids produced with T 1 COMT-downregulated switchgrass as the female parent were taller, produced more tillers, and produced 63% more biomass compared with those produced using the field selected accession as the female parent. Transgene status (presence or absence of transgene) influenced both growth and cell wall traits. T 2 transgenic hybrids were 7% shorter 80 days after sowing and produced 43% less biomass than non-transgenic null-segregant hybrids. Cell wall-related differences included lower lignin content, reduced syringyl-to-guaiacyl (S/G) lignin monomer ratio, and a 12% increase in total sugar release in the T 2 transgenic hybrids compared to non-transgenic null segregants. This is the first study to evaluate the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits. Lastly, our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.« less

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. ©AlphaMed Press.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M−/− hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M−/− hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M−/− hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells. PMID:26285657

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells.

PubMed

El-Hoss, Jad; Sullivan, Kate; Cheng, Tegan; Yu, Nicole Y C; Bobyn, Justin D; Peacock, Lauren; Mikulec, Kathy; Baldock, Paul; Alexander, Ian E; Schindeler, Aaron; Little, David G

2012-01-01

Neurofibromatosis type 1 (NF1) is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue-specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1(flox/flox) and Nf1(flox/-) mice. The effects of the presence of Nf1(null) cells were extensively examined. Cultured Nf1(null)-committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with recombinant bone morphogenetic protein-2 (rhBMP-2) treatment. Similarly, Nf1(null)-inducible osteoprogenitors obtained from Nf1 MyoDnull mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1(flox/flox) and Nf1(flox/-) mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1(flox/flox) and Nf1(flox/-) mice. Histological analyses showed invasion of the Nf1(null) fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upward of 10-fold in Nf1(null) fractures and bromodeoxyuridine (BrdU) staining in closed fractures showed increased numbers of proliferating cells. In Nf1(null) fractures, tartrate-resistant acid phosphatase-positive (TRAP+) cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds. Copyright © 2012 American Society for Bone and Mineral Research.

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity.

PubMed

Meshulam, Tova; Breen, Michael R; Liu, Libin; Parton, Robert G; Pilch, Paul F

2011-08-01

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains.

Caveolins/caveolae protect adipocytes from fatty acid-mediated lipotoxicity

PubMed Central

Meshulam, Tova; Breen, Michael R.; Liu, Libin; Parton, Robert G.; Pilch, Paul F.

2011-01-01

Mice and humans lacking functional caveolae are dyslipidemic and have reduced fat stores and smaller fat cells. To test the role of caveolins/caveolae in maintaining lipid stores and adipocyte integrity, we compared lipolysis in caveolin-1 (Cav1)-null fat cells to that in cells reconstituted for caveolae by caveolin-1 re-expression. We find that the Cav1-null cells have a modestly enhanced rate of lipolysis and reduced cellular integrity compared with reconstituted cells as determined by the release of lipid metabolites and lactic dehydrogenase, respectively, into the media. There are no apparent differences in the levels of lipolytic enzymes or hormonally stimulated phosphorylation events in the two cell lines. In addition, acute fasting, which dramatically raises circulating fatty acid levels in vivo, causes a significant upregulation of caveolar protein constituents. These results are consistent with the hypothesis that caveolae protect fat cells from the lipotoxic effects of elevated levels fatty acids, which are weak detergents at physiological pH, by virtue of the property of caveolae to form detergent-resistant membrane domains. PMID:21652731

Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

NASA Technical Reports Server (NTRS)

Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

2000-01-01

Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

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

Bassuk, James; Lendvay, Thomas S.; Sweet, Robert

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functionalmore » protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo.To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.« less

Terrestrial Planet Finder cryogenic delay line development

NASA Technical Reports Server (NTRS)

Smythe, Robert F.; Swain, Mark R.; Alvarez-Salazar, Oscar; Moore, James D.

2004-01-01

Delay lines provide the path-length compensation that makes the measurement of interference fringes possible. When used for nulling interferometry, the delay line must control path-lengths so that the null is stable and controlled throughout the measurement. We report on a low noise, low disturbance, and high bandwidth optical delay line capable of meeting the TPF interferometer optical path length control requirements at cryogenic temperatures.

Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

PubMed

Xin, Jige; Yang, Huaqiang; Fan, Nana; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Zhao, Yu; Li, Xiaoping; Song, Jun; Yang, Yi; Zou, Qingjian; Yan, Quanmei; Zeng, Yangzhi; Lai, Liangxue

2013-01-01

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

PubMed

Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

1999-08-01

Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

PubMed Central

Keighren, Margaret A.; Flockhart, Jean H.

2016-01-01

ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

Peripheral Frequency of CD4+ CD28− Cells in Acute Ischemic Stroke

PubMed Central

Tuttolomondo, Antonino; Pecoraro, Rosaria; Casuccio, Alessandra; Di Raimondo, Domenico; Buttà, Carmelo; Clemente, Giuseppe; Corte, Vittoriano della; Guggino, Giuliana; Arnao, Valentina; Maida, Carlo; Simonetta, Irene; Maugeri, Rosario; Squatrito, Rosario; Pinto, Antonio

2015-01-01

Abstract CD4+ CD28− T cells also called CD28 null cells have been reported as increased in the clinical setting of acute coronary syndrome. Only 2 studies previously analyzed peripheral frequency of CD28 null cells in subjects with acute ischemic stroke but, to our knowledge, peripheral frequency of CD28 null cells in each TOAST subtype of ischemic stroke has never been evaluated. We hypothesized that CD4+ cells and, in particular, the CD28 null cell subset could show a different degree of peripheral percentage in subjects with acute ischemic stroke in relation to clinical subtype and severity of ischemic stroke. The aim of our study was to analyze peripheral frequency of CD28 null cells in subjects with acute ischemic stroke in relation to TOAST diagnostic subtype, and to evaluate their relationship with scores of clinical severity of acute ischemic stroke, and their predictive role in the diagnosis of acute ischemic stroke and diagnostic subtype We enrolled 98 consecutive subjects admitted to our recruitment wards with a diagnosis of ischemic stroke. As controls we enrolled 66 hospitalized patients without a diagnosis of acute ischemic stroke. Peripheral frequency of CD4+ and CD28 null cells has been evaluated with a FACS Calibur flow cytometer. Subjects with acute ischemic stroke had a significantly higher peripheral frequency of CD4+ cells and CD28 null cells compared to control subjects without acute ischemic stroke. Subjects with cardioembolic stroke had a significantly higher peripheral frequency of CD4+ cells and CD28 null cells compared to subjects with other TOAST subtypes. We observed a significant relationship between CD28 null cells peripheral percentage and Scandinavian Stroke Scale and NIHSS scores. ROC curve analysis showed that CD28 null cell percentage may be useful to differentiate between stroke subtypes. These findings seem suggest a possible role for a T-cell component also in acute ischemic stroke clinical setting showing a different peripheral frequency of CD28 null cells in relation of each TOAST subtype of stroke. PMID:25997053

Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death.

PubMed

Conforti, Paola; Camnasio, Stefano; Mutti, Cesare; Valenza, Marta; Thompson, Morgan; Fossale, Elisa; Zeitlin, Scott; MacDonald, Marcy E; Zuccato, Chiara; Cattaneo, Elena

2013-02-01

Huntington's disease (HD) is a neurodegenerative disorder that affects muscle coordination and diminishes cognitive abilities. The genetic basis of the disease is an expansion of CAG repeats in the Huntingtin (Htt) gene. Here we aimed to generate a series of mouse neural stem (NS) cell lines that carried varying numbers of CAG repeats in the mouse Htt gene (Hdh CAG knock-in NS cells) or that had Hdh null alleles (Hdh knock-out NS cells). Towards this end, Hdh CAG knock-in mouse ES cell lines that carried an Htt gene with 20, 50, 111, or 140 CAG repeats or that were Htt null were neuralized and converted into self-renewing NS cells. The resulting NS cell lines were immunopositive for the neural stem cell markers NESTIN, SOX2, and BLBP and had similar proliferative rates and cell cycle distributions. After 14 days in vitro, wild-type NS cells gave rise to cultures composed of 70% MAP2(+) neurons and 30% GFAP(+) astrocytes. In contrast, NS cells with expanded CAG repeats underwent neuronal cell death, with only 38%±15% of the MAP2(+) cells remaining at the end of the differentiation period. Cell death was verified by increased caspase 3/7 activity on day 14 of the neuronal differentiation protocol. Interestingly, Hdh knock-out NS cells treated using the same neuronal differentiation protocol showed a dramatic increase in the number of GFAP(+) cells on day 14 (61%±20% versus 24%±10% in controls), and a massive decrease of MAP2(+) neurons (30%±11% versus 64%±17% in controls). Both Hdh CAG knock-in NS cells and Hdh knock-out NS cells showed reduced levels of Bdnf mRNA during neuronal differentiation, in agreement with data obtained previously in HD mouse models and in post-mortem brain samples from HD patients. We concluded that Hdh CAG knock-in and Hdh knock-out NS cells have potential as tools for investigating the roles of normal and mutant HTT in differentiated neurons and glial cells of the brain. Copyright © 2012 Elsevier Inc. All rights reserved.

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials.

PubMed

Lin, Ann; Giuliano, Christopher J; Sayles, Nicole M; Sheltzer, Jason M

2017-03-24

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.

Caspase-1 from Human Myeloid-Derived Suppressor Cells Can Promote T Cell-Independent Tumor Proliferation.

PubMed

Zeng, Qi; Fu, Juan; Korrer, Michael; Gorbounov, Mikhail; Murray, Peter J; Pardoll, Drew; Masica, David L; Kim, Young J

2018-05-01

Immunosuppressive myeloid-derived suppressive cells (MDSCs) are characterized by their phenotypic and functional heterogeneity. To better define their T cell-independent functions within the tumor, sorted monocytic CD14 + CD11b + HLA-DR low/- MDSCs (mMDSC) from squamous cell carcinoma patients showed upregulated caspase-1 activity, which was associated with increased IL1β and IL18 expression. In vitro studies demonstrated that mMDSCs promoted caspase-1-dependent proliferation of multiple squamous carcinoma cell lines in both human and murine systems. In vivo , growth rates of B16, MOC1, and Panc02 were significantly blunted in chimeric mice adoptively transferred with caspase-1 null bone marrow cells under T cell-depleted conditions. Adoptive transfer of wild-type Gr-1 + CD11b + MDSCs from tumor-bearing mice reversed this antitumor response, whereas caspase-1 inhibiting thalidomide-treated MDSCs phenocopied the antitumor response found in caspase-1 null mice. We further hypothesized that MDSC caspase-1 activity could promote tumor-intrinsic MyD88-dependent carcinogenesis. In mice with wild-type caspase-1, MyD88-silenced tumors displayed reduced growth rate, but in chimeric mice with caspase-1 null bone marrow cells, MyD88-silenced tumors did not display differential tumor growth rate. When we queried the TCGA database, we found that caspase-1 expression is correlated with overall survival in squamous cell carcinoma patients. Taken together, our findings demonstrated that caspase-1 in MDSCs is a direct T cell-independent mediator of tumor proliferation. Cancer Immunol Res; 6(5); 566-77. ©2018 AACR . ©2018 American Association for Cancer Research.

Protein and quality characterization of complete and partial near isogenic lines of waxy wheat

USDA-ARS?s Scientific Manuscript database

The objective of this study was to evaluate protein composition and its effects on flour quality and physical dough test parameters using waxy wheat near-isogenic lines. Partial waxy (single and double nulls) and waxy (null at all three waxy loci, Wx-A1, Wx-B1, and Wx-D1) lines of N11 set (bread whe...

Isolation of a novel chronic lymphocytic leukemic (CLL) cell line and development of an in vivo mouse model of CLL.

PubMed

Kellner, Joshua; Wierda, William; Shpall, Elizabeth; Keating, Michael; McNiece, Ian

2016-01-01

Leukemic cell lines have become important tools for studies of disease providing a monoclonal cell population that can be extensively expanded in vitro while preserving leukemic cellular characteristics. However, studies of chronic lymphocytic leukemia (CLL) have been impeded in part by the lack of continuous human cell lines. CLL cells have a high spontaneous apoptosis rate in vitro and exhibit minimal proliferation in xenograft models. Therefore, there is a need for development of primary CLL cell lines and we describe the isolation of such a line from the bone marrow of a CLL patient (17p deletion and TP53 mutation) which has been in long term culture for more than 12 months with continuous proliferation. The CLL cell line (termed MDA-BM5) which was generated in vitro with continuous co-culture on autologous stromal cells is CD19+CD5+ and shows an identical pattern of somatic hypermutation as determined in the patient's bone marrow (BM), confirming the origin of the cells from the original CLL clone. MDA-BM5 cells were readily transplantable in NOD/SCID gamma null mice (NSG) with disease developing in the BM, liver and spleen. BM cells from quaternary serial transplantation in NSG mice demonstrated the presence of CD19+CD5+ cells with Ig restricted to lambda which is consistent with the original patient cells. These studies describe a new CLL cell line from a patient with del(17p) that provides a unique model for in vitro and in vivo studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Magnetohydrodynamic Simulation of Magnetic Null-point Reconnections in NOAA AR 12192, Initiated with an Extrapolated Non-force-free Field

NASA Astrophysics Data System (ADS)

Prasad, A.; Bhattacharyya, R.; Hu, Qiang; Kumar, Sanjay; Nayak, Sushree S.

2018-06-01

The magnetohydrodynamics of the solar corona is simulated numerically. The simulation is initialized with an extrapolated non-force-free magnetic field using the vector magnetogram of the active region NOAA 12192, which was obtained from the solar photosphere. Particularly, we focus on the magnetic reconnections (MRs) occurring close to a magnetic null point that resulted in the appearance of circular chromospheric flare ribbons on 2014 October 24 around 21:21 UT, after the peak of an X3.1 flare. The extrapolated field lines show the presence of the three-dimensional (3D) null near one of the polarity-inversion lines—where the flare was observed. In the subsequent numerical simulation, we find MRs occurring near the null point, where the magnetic field lines from the fan plane of the 3D null form a X-type configuration with underlying arcade field lines. The footpoints of the dome-shaped field lines, inherent to the 3D null, show high gradients of the squashing factor. We find slipping reconnections at these quasi-separatrix layers, which are co-located with the post-flare circular brightening observed at chromospheric heights. This demonstrates the viability of the initial non-force-free field, along with the dynamics it initiates. Moreover, the initial field and its simulated evolution are found to be devoid of any flux rope, which is congruent with the confined nature of the flare.

Decidual activin: its role in the apoptotic process and its regulation by prolactin.

PubMed

Tessier, Christian; Prigent-Tessier, Anne; Bao, Lei; Telleria, Carlos M; Ferguson-Gottschall, Susan; Gibori, Gil B; Gu, Yan; Bowen-Shauver, Jennifer M; Horseman, Nelson D; Gibori, Geula

2003-05-01

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

DTIC Science & Technology

2006-06-01

transfected into 184A1-Babe and 184A1-GSE22 cells at a 1:1 ratio with the promoterless Renilla reporter plasmid (pRL- Null). Following a 48h incubation...reporter luminescence to the Renilla reporter luminescence. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 184A1-Babe p15 pGL3- Basic 184A1-Babe p15 pGL3- 3396

Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer

DTIC Science & Technology

2016-09-01

in this progress report: p53 triple-negative breast cancer subtypes gene expression somatic cell genetics CRISPR /Cas 3. ACCOMPLISHMENTS Major...report, we described the creation of an isogenic p53 mutant TNBC cell line panel using CRISPR /Cas-mediated genome editing8 and the resultant...LOF null state. To validate that mutant p53 is directly responsible for this altered transcription, we will use the same CRISPR -mediated genome

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

PubMed

Miller, Jason R; Koren, Sergey; Dilley, Kari A; Puri, Vinita; Brown, David M; Harkins, Derek M; Thibaud-Nissen, Françoise; Rosen, Benjamin; Chen, Xiao-Guang; Tu, Zhijian; Sharakhov, Igor V; Sharakhova, Maria V; Sebra, Robert; Stockwell, Timothy B; Bergman, Nicholas H; Sutton, Granger G; Phillippy, Adam M; Piermarini, Peter M; Shabman, Reed S

2018-03-01

The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

PubMed

Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

Anti-tumor effects of suberoylanilide hydroxamic acid on Epstein–Barr virus-associated T cell and natural killer cell lymphoma

PubMed Central

Siddiquey, Mohammed NA; Nakagawa, Hikaru; Iwata, Seiko; Kanazawa, Tetsuhiro; Suzuki, Michio; Imadome, Ken-Ichi; Fujiwara, Shigeyoshi; Goshima, Fumi; Murata, Takayuki; Kimura, Hiroshi

2014-01-01

The ubiquitous Epstein–Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells and is associated with various lymphoid malignancies. Recent studies have reported that histone deacetylase (HDAC) inhibitors exert anticancer effects against various tumor cells. In the present study, we have evaluated both the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on EBV-positive and EBV-negative T and NK lymphoma cells. Several EBV-positive and EBV-negative T and NK cell lines were treated with various concentrations of SAHA. SAHA suppressed the proliferation of T and NK cell lines, although no significant difference was observed between EBV-positive and EBV-negative cell lines. SAHA induced apoptosis and/or cell cycle arrest in several T and NK cell lines. In addition, SAHA increased the expression of EBV-lytic genes and decreased the expression of EBV-latent genes. Next, EBV-positive NK cell lymphoma cells were subcutaneously inoculated into severely immunodeficient NOD/Shi-scid/IL-2Rγnull mice, and then SAHA was administered intraperitoneally. SAHA inhibited tumor progression and metastasis in the murine xenograft model. SAHA displayed a marked suppressive effect against EBV-associated T and NK cell lymphomas through either induction of apoptosis or cell cycle arrest, and may represent an alternative treatment option. PMID:24712440

Numerical simulations of sheared magnetic lines at the solar null line

NASA Astrophysics Data System (ADS)

Kuźma, B.; Murawski, K.; Solov'ev, A.

2015-05-01

Aims: We perform numerical simulations of sheared magnetic lines at the magnetic null line configuration of two magnetic arcades that are settled in a gravitationally stratified and magnetically confined solar corona. Methods: We developed a general analytical model of a 2.5D solar atmospheric structure. As a particular application of this model, we adopted it for the curved magnetic field lines with an inverted Y shape that compose the null line above two magnetic arcades, which are embedded in the solar atmosphere that is specified by the realistic temperature distribution. The physical system is described by 2.5D magnetohydrodynamic equations that are numerically solved by the FLASH code. Results: The magnetic field line shearing, implemented about 200 km below the transition region, results in Alfvén and magnetoacoustic waves that are able to penetrate solar coronal regions above the magnetic null line. As a result of the coupling of these waves, partial reflection from the transition region and scattering from inhomogeneous regions the Alfvén waves experience fast attenuation on time scales comparable to their wave periods, and the physical system relaxes in time. The attenuation time grows with the large amplitude and characteristic growing time of the shearing. Conclusions: By having chosen a different magnetic flux function, the analytical model we devised can be adopted to derive equilibrium conditions for a diversity of 2.5D magnetic structures in the solar atmosphere. Movie associated to Fig. 5 is available in electronic form at http://www.aanda.org

Glucosylceramide is Critical for Cell-Type Differentiation and Organogenesis, but not for Cell Viability in Arabidopsis

PubMed Central

Msanne, Joseph; Chen, Ming; Luttgeharm, Kyle D.; Bradley, Amanda M.; Mays, Elizabeth S.; Paper, Janet M.; Boyle, Daniel L.; Cahoon, Rebecca E.; Schrick, Kathrin; Cahoon, Edgar B.

2015-01-01

Summary Glucosylceramides (GlcCer), glucose-conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryote cells. Yet, the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi-organ eukaryotes. To address this, we examined Arabidopsis lines lacking or deficient in GlcCer by insertional disruption or by RNAi suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated “gcs-1”) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs-1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild-type plants. However, gcs-1 calli, in contrast to wild-type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ-specific cell differentiation, calli from gcs-1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs-1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild-type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild-type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell-type differentiation and organogenesis, and plant cells produce GlcCer amounts in excess of that required for normal development. PMID:26313010

Vaccination with a Leishmania infantum HSP70-II null mutant confers long-term protective immunity against Leishmania major infection in two mice models

PubMed Central

Solana, José Carlos; Ramírez, Laura; Corvo, Laura; de Oliveira, Camila Indiani; Barral-Netto, Manoel; Requena, José María

2017-01-01

Background The immunization with genetically attenuated Leishmania cell lines has been associated to the induction of memory and effector T cell responses against Leishmania able to control subsequent challenges. A Leishmania infantum null mutant for the HSP70-II genes has been described, possessing a non-virulent phenotype. Methodology/Principal findings The L. infantum attenuated parasites (LiΔHSP70-II) were inoculated in BALB/c (intravenously and subcutaneously) and C57BL/6 (subcutaneously) mice. An asymptomatic infection was generated and parasites diminished progressively to become undetectable in most of the analyzed organs. However, inoculation resulted in the long-term induction of parasite specific IFN-γ responses able to control the disease caused by a challenge of L. major infective promastigotes. BALB/c susceptible mice showed very low lesion development and a drastic decrease in parasite burdens in the lymph nodes draining the site of infection and internal organs. C57BL/6 mice did not show clinical manifestation of disease, correlated to the rapid migration of Leishmania specific IFN-γ producing T cells to the site of infection. Conclusion/Significance Inoculation of the LiΔHSP70-II attenuated line activates mammalian immune system for inducing moderate pro-inflammatory responses. These responses are able to confer long-term protection in mice against the infection of L. major virulent parasites. PMID:28558043

The cell-specific pattern of cholecystokinin peptides in endocrine cells versus neurons is governed by the expression of prohormone convertases 1/3, 2, and 5/6.

PubMed

Rehfeld, Jens F; Bundgaard, Jens R; Hannibal, Jens; Zhu, Xiaorong; Norrbom, Christina; Steiner, Donald F; Friis-Hansen, Lennart

2008-04-01

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendocrine prohormone convertases (PC) 1/3, PC2, and PC5/6 by measurement of proCCK, processing intermediates and bioactive, alpha-amidated, and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of alpha-amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK cells in wild-type mice express PC1/3 but not PC2. In contrast, cerebral CCK neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of PCs.

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials

PubMed Central

Lin, Ann; Giuliano, Christopher J; Sayles, Nicole M; Sheltzer, Jason M

2017-01-01

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied. DOI: http://dx.doi.org/10.7554/eLife.24179.001 PMID:28337968

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

PubMed

Lee, S; Parent, C A; Insall, R; Firtel, R A

1999-09-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Hypersensitivities for Acetaldehyde and Other Agents among Cancer Cells Null for Clinically Relevant Fanconi Anemia Genes

PubMed Central

Ghosh, Soma; Sur, Surojit; Yerram, Sashidhar R.; Rago, Carlo; Bhunia, Anil K.; Hossain, M. Zulfiquer; Paun, Bogdan C.; Ren, Yunzhao R.; Iacobuzio-Donahue, Christine A.; Azad, Nilofer A.; Kern, Scott E.

2014-01-01

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities. PMID:24200853

Altered Expression of a Malate-Permeable Anion Channel, OsALMT4, Disrupts Mineral Nutrition1[OPEN

PubMed Central

Delhaize, Emmanuel

2017-01-01

Aluminum-activated malate transporters (ALMTs) form a family of anion channels in plants, but little is known about most of its members. This study examined the function of OsALMT4 from rice (Oryza sativa). We show that OsALMT4 is expressed in roots and shoots and that the OsALMT4 protein localizes to the plasma membrane. Transgenic rice lines overexpressing (OX) OsALMT4 released malate from the roots constitutively and had 2-fold higher malate concentrations in the xylem sap than nulls, indicating greater concentrations of malate in the apoplast. OX lines developed brown necrotic spots on the leaves that did not appear on nulls. These symptoms were not associated with altered concentrations of any mineral element in the leaves, although the OX lines had higher concentrations of Mn and B in their grain compared with nulls. While total leaf Mn concentrations were not different between the OX and null lines, Mn concentrations in the apoplast were greater in the OX plants. The OX lines also displayed increased expression of Mn transporters and were more sensitive to Mn toxicity than null plants. We showed that the growth of wild-type rice was unaffected by 100 µm Mn in hydroponics but, when combined with 1 mm malate, this concentration inhibited growth. We conclude that increasing OsALMT4 expression affected malate efflux and compartmentation within the tissues, which increased Mn concentrations in the apoplast of leaves and induced the toxicity symptoms. This study reveals new links between malate transport and mineral nutrition. PMID:29101278

Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1

PubMed Central

Jang, Hyeung-Jin; Kokrashvili, Zaza; Theodorakis, Michael J.; Carlson, Olga D.; Kim, Byung-Joon; Zhou, Jie; Kim, Hyeon Ho; Xu, Xiangru; Chan, Sic L.; Juhaszova, Magdalena; Bernier, Michel; Mosinger, Bedrich; Margolskee, Robert F.; Egan, Josephine M.

2007-01-01

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express α-gustducin. Ingestion of glucose by α-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from α-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses α-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for α-gustducin. We conclude that L cells of the gut “taste” glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut “taste cells” may provide an important treatment for obesity, diabetes and abnormal gut motility. PMID:17724330

Immunological role of CD4+CD28null T lymphocytes, natural killer cells, and interferon-gamma in pediatric patients with sickle cell disease: relation to disease severity and response to therapy.

PubMed

ElAlfy, Mohsen Saleh; Adly, Amira Abdel Moneam; Ebeid, Fatma Soliman ElSayed; Eissa, Deena Samir; Ismail, Eman Abdel Rahman; Mohammed, Yasser Hassan; Ahmed, Manar Elsayed; Saad, Aya Sayed

2018-06-20

Sickle cell disease (SCD) is associated with alterations in immune phenotypes. CD4 + CD28 null T lymphocytes have pro-inflammatory functions and are linked to vascular diseases. To assess the percentage of CD4 + CD28 null T lymphocytes, natural killer cells (NK), and IFN-gamma levels, we compared 40 children and adolescents with SCD with 40 healthy controls and evaluated their relation to disease severity and response to therapy. Patients with SCD steady state were studied, focusing on history of frequent vaso-occlusive crisis, hydroxyurea therapy, and IFN-gamma levels. Analysis of CD4 + CD28 null T lymphocytes and NK cells was done by flow cytometry. Liver and cardiac iron overload were assessed. CD4 + CD28 null T lymphocytes, NK cells, and IFN-gamma levels were significantly higher in patients than controls. Patients with history of frequent vaso-occlusive crisis and those with vascular complications had higher percentage of CD4 + CD28 null T lymphocytes and IFN-gamma while levels were significantly lower among hydroxyurea-treated patients. CD4 + CD28 null T lymphocytes were positively correlated to transfusional iron input while these cells and IFN-gamma were negatively correlated to cardiac T2* and duration of hydroxyurea therapy. NK cells were correlated to HbS and indirect bilirubin. Increased expression of CD4 + CD28 null T lymphocytes highlights their role in immune dysfunction and pathophysiology of SCD complications.

The von Hippel-Lindau protein sensitizes renal carcinoma cells to apoptotic stimuli through stabilization of BIM(EL).

PubMed

Guo, Y; Schoell, M C; Freeman, R S

2009-04-23

von Hippel-Lindau (VHL) disease is caused by germ-line mutations in the VHL tumor suppressor gene and is the most common cause of inherited renal cell carcinoma (RCC). Mutations in the VHL gene also occur in a large majority of sporadic cases of clear-cell RCC, which have high intrinsic resistance to chemotherapy and radiotherapy. Here we show that VHL-deficient RCC cells express lower levels of the proapoptotic Bcl-2 family protein BIM(EL) and are more resistant to etoposide and UV radiation-induced death compared to the same cells stably expressing the wild-type VHL protein (pVHL). Reintroducing pVHL into VHL-null cells increased the half-life of BIM(EL) protein without affecting its mRNA expression, and overexpressing pVHL inhibited BIM(EL) polyubiquitination. Suppressing pVHL expression with RNA interference resulted in a decrease in BIM(EL) protein and a corresponding decrease in the sensitivity of RCC cells to apoptotic stimuli. Directly inhibiting BIM(EL) expression in pVHL-expressing RCC cells caused a similar decrease in cell death. These results demonstrate that pVHL acts to promote BIM(EL) protein stability in RCC cells, and that destabilization of BIM(EL) in the absence of pVHL contributes to the increased resistance of VHL-null RCC cells to certain apoptotic stimuli.

The prolyl isomerase Pin1 modulates development of CD8+ cDC in mice.

PubMed

Barberi, Theresa J; Dunkle, Alexis; He, You-Wen; Racioppi, Luigi; Means, Anthony R

2012-01-01

Pin1 has previously been described to regulate cells that participate in both innate and adaptive immunity. Thus far, however, no role for Pin1 has been described in modulating conventional dendritic cells, innate antigen presenting cells that potently activate naïve T cells, thereby bridging innate and adaptive immune responses. When challenged with LPS, Pin1-null mice failed to accumulate spleen conventional dendritic cells (cDC). Analysis of steady-state spleen DC populations revealed that Pin1-null mice had fewer CD8+ cDC. This defect was recapitulated by culturing Pin1-null bone marrow with the DC-instructive cytokine Flt3 Ligand. Additionally, injection of Flt3 Ligand for 9 days failed to induce robust expansion of CD8+ cDC in Pin1-null mice. Upon infection with Listeria monocytogenes, Pin1-null mice were defective in stimulating proliferation of adoptively transferred WT CD8+ T cells, suggesting that decreases in Pin1 null CD8+ cDC may affect T cell responses to infection in vivo. Finally, upon analyzing expression of proteins involved in DC development, elevated expression of PU.1 was detected in Pin1-null cells, which resulted from an increase in PU.1 protein half-life. We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity.

PTEN loss promotes intratumoral androgen synthesis and tumor microenvironment remodeling via aberrant activation of RUNX2 in castration-resistant prostate cancer

PubMed Central

Yang, Yinhui; Bai, Yang; He, Yundong; Zhao, Yu; Chen, Jiaxiang; Ma, Linlin; Pan, Yunqian; Hinten, Michael; Zhang, Jun; Karnes, R. Jeffrey; Kohli, Manish; Westendorf, Jennifer J.; Li, Benyi; Zhu, Runzhi; Huang, Haojie; Xu, Wanhai

2018-01-01

Purpose Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR)reactivation and anti-androgen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood. Experimental Design The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human PCa cell lines. Pten knockout mice were employed to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivo. Results We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null PCa cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivo. Pten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9. Conclusions Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null PCa including CRPC. PMID:29167276

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

PubMed

Hsu, Jessie Hao-Ru; Hubbell-Engler, Benjamin; Adelmant, Guillaume; Huang, Jialiang; Joyce, Cailin E; Vazquez, Francisca; Weir, Barbara A; Montgomery, Philip; Tsherniak, Aviad; Giacomelli, Andrew O; Perry, Jennifer A; Trowbridge, Jennifer; Fujiwara, Yuko; Cowley, Glenn S; Xie, Huafeng; Kim, Woojin; Novina, Carl D; Hahn, William C; Marto, Jarrod A; Orkin, Stuart H

2017-09-01

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR . ©2017 American Association for Cancer Research.

Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

PubMed

Ghosh, Soma; Sur, Surojit; Yerram, Sashidhar R; Rago, Carlo; Bhunia, Anil K; Hossain, M Zulfiquer; Paun, Bogdan C; Ren, Yunzhao R; Iacobuzio-Donahue, Christine A; Azad, Nilofer A; Kern, Scott E

2014-01-01

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3

PubMed Central

Zhou, Gaofeng; Ryan, Peter R.

2014-01-01

Malate and citrate efflux from root apices is a mechanism of Al3+ tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al3+-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al3+ tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al3+-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al3+-activated citrate efflux from root apices and greater tolerance to Al3+ toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al3+ tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al3+ tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al3+ tolerance of an important crop species. PMID:24692647

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

PubMed

Heuchel, R; Radtke, F; Georgiev, O; Stark, G; Aguet, M; Schaffner, W

1994-06-15

We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

PubMed Central

Lee, Susan; Parent, Carole A.; Insall, Robert; Firtel, Richard A.

1999-01-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation. PMID:10473630

Induction of CaSR expression circumvents the molecular features of malignant CaSR null colon cancer cells.

PubMed

Singh, Navneet; Chakrabarty, Subhas

2013-11-15

We recently reported on the isolation and characterization of calcium sensing receptor (CaSR) null human colon cancer cells (Singh et al., Int J Cancer 2013; 132: 1996-2005). CaSR null cells possess a myriad of molecular features that are linked to a highly malignant and drug resistant phenotype of colon cancer. The CaSR null phenotype can be maintained in defined human embryonic stem cell culture medium. We now show that the CaSR null cells can be induced to differentiate in conventional culture medium, regained the expression of CaSR with a concurrent reversal of the cellular and molecular features associated with the null phenotype. These features include cellular morphology, expression of colon cancer stem cell markers, expression of survivin and thymidylate synthase and sensitivity to fluorouracil. Other features include the expression of epithelial mesenchymal transition linked molecules and transcription factors, oncogenic miRNAs and tumor suppressive molecule and miRNA. With the exception of cancer stem cell markers, the reversal of molecular features, upon the induction of CaSR expression, is directly linked to the expression and function of CaSR because blocking CaSR induction by shRNA circumvented such reversal. We further report that methylation and demethylation of the CaSR gene promoter underlie CaSR expression. Due to the malignant nature of the CaSR null cells, inclusion of the CaSR null phenotype in disease management may improve on the mortality of this disease. Because CaSR is a robust promoter of differentiation and mediates its action through diverse mechanisms and pathways, inactivation of CaSR may serve as a new paradigm in colon carcinogenesis. Copyright © 2013 UICC.

Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

PubMed

Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

2015-01-01

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

Metallothionein blocks oxidative DNA damage induced by acute inorganic arsenic exposure

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

Qu, Wei, E-mail: qu@niehs.nih.gov; Waalkes, Michael P.

We studied how protein metallothionein (MT) impacts arsenic-induced oxidative DNA damage (ODD) using cells that poorly express MT (MT-I/II double knockout embryonic cells; called MT-null cells) and wild-type (WT) MT competent cells. Arsenic (as NaAsO{sub 2}) was less cytolethal over 24 h in WT cells (LC{sub 50} = 11.0 ± 1.3 μM; mean ± SEM) than in MT-null cells (LC{sub 50} = 5.6 ± 1.2 μM). ODD was measured by the immuno-spin trapping method. Arsenic (1 or 5 μM; 24 h) induced much less ODD in WT cells (121% and 141% of control, respectively) than in MT-null cells (202% andmore » 260%). In WT cells arsenic caused concentration-dependent increases in MT expression (transcript and protein), and in the metal-responsive transcription factor-1 (MTF-1), which is required to induce the MT gene. In contrast, basal MT levels were not detectable in MT-null cells and unaltered by arsenic exposure. Transfection of MT-I gene into the MT-null cells markedly reduced arsenic-induced ODD levels. The transport genes, Abcc1 and Abcc2 were increased by arsenic in WT cells but either showed no or very limited increases in MT-null cells. Arsenic caused increases in oxidant stress defense genes HO-1 and GSTα2 in both WT and MT-null cells, but to much higher levels in WT cells. WT cells appear more adept at activating metal transport systems and oxidant response genes, although the role of MT in these responses is unclear. Overall, MT protects against arsenic-induced ODD in MT competent cells by potential sequestration of scavenging oxidant radicals and/or arsenic. - Highlights: • Metallothionein blocks arsenic toxicity. • Metallothionein reduces arsenic-induced DNA damage. • Metallothionein may bind arsenic or radicals produced by arsenic.« less

Mitogen- and Stress-Activated Protein Kinase 1 Regulates Status Epilepticus-Evoked Cell Death in the Hippocampus

PubMed Central

Choi, Yun-Sik; Horning, Paul; Aten, Sydney; Karelina, Kate; Alzate-Correa, Diego; Arthur, J. Simon C.; Hoyt, Kari R.; Obrietan, Karl

2017-01-01

Mitogen-activated protein kinase (MAPK) signaling has been implicated in a wide range of neuronal processes, including development, plasticity, and viability. One of the principal downstream targets of both the extracellular signal-regulated kinase/MAPK pathway and the p38 MAPK pathway is Mitogen- and Stress-activated protein Kinase 1 (MSK1). Here, we sought to understand the role that MSK1 plays in neuroprotection against excitotoxic stimulation in the hippocampus. To this end, we utilized immunohistochemical labeling, a MSK1 null mouse line, cell viability assays, and array-based profiling approaches. Initially, we show that MSK1 is broadly expressed within the major neuronal cell layers of the hippocampus and that status epilepticus drives acute induction of MSK1 activation. In response to the status epilepticus paradigm, MSK1 KO mice exhibited a striking increase in vulnerability to pilocarpine-evoked cell death within the CA1 and CA3 cell layers. Further, cultured MSK1 null neurons exhibited a heighted level of N-methyl-D-aspartate-evoked excitotoxicity relative to wild-type neurons, as assessed using the lactate dehydrogenase assay. Given these findings, we examined the hippocampal transcriptional profile of MSK1 null mice. Affymetrix array profiling revealed that MSK1 deletion led to the significant (>1.25-fold) downregulation of 130 genes and an upregulation of 145 genes. Notably, functional analysis indicated that a subset of these genes contribute to neuroprotective signaling networks. Together, these data provide important new insights into the mechanism by which the MAPK/MSK1 signaling cassette confers neuroprotection against excitotoxic insults. Approaches designed to upregulate or mimic the functional effects of MSK1 may prove beneficial against an array of degenerative processes resulting from excitotoxic insults. PMID:28870089

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

PubMed Central

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. PMID:27856772

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

PubMed

Soni, Shivani; Bala, Shashi; Hanspal, Manjit

2008-01-01

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer

PubMed Central

Li, Jie; Koike, Junichi; Kugoh, Hiroyuki; Arita, Michitsune; Ohhira, Takahito; Kikuchi, Yoshinori; Funahashi, Kimihiko; Takamatsu, Ken; Boland, C. Richard; Koi, Minoru; Hemmi, Hiromichi

2013-01-01

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells. PMID:22343000

Discovery of ALK-PTPN3 gene fusion from human non-small cell lung carcinoma cell line using next generation RNA sequencing.

PubMed

Jung, Yeonjoo; Kim, Pora; Jung, Yeonhwa; Keum, Juhee; Kim, Soon-Nam; Choi, Yong Soo; Do, In-Gu; Lee, Jinseon; Choi, So-Jung; Kim, Sujin; Lee, Jong-Eun; Kim, Jhingook; Lee, Sanghyuk; Kim, Jaesang

2012-06-01

An increasing number of chromosomal aberrations is being identified in solid tumors providing novel biomarkers for various types of cancer and new insights into the mechanisms of carcinogenesis. We applied next generation sequencing technique to analyze the transcriptome of the non-small cell lung carcinoma (NSCLC) cell line H2228 and discovered a fusion transcript composed of multiple exons of ALK (anaplastic lymphoma receptor tyrosine kinase) and PTPN3 (protein tyrosine phosphatase, nonreceptor Type 3). Detailed analysis of the genomic structure revealed that a portion of genomic region encompassing Exons 10 and 11 of ALK has been translocated into the intronic region between Exons 2 and 3 of PTPN3. The key net result appears to be the null mutation of one allele of PTPN3, a gene with tumor suppressor activity. Consistently, ectopic expression of PTPN3 in NSCLC cell lines led to inhibition of colony formation. Our study confirms the utility of next generation sequencing as a tool for the discovery of somatic mutations and has led to the identification of a novel mutation in NSCLC that may be of diagnostic, prognostic, and therapeutic importance. Copyright © 2012 Wiley Periodicals, Inc.

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes.

PubMed

deHart, Gregory W; Healy, Kevin E; Jones, Jonathan C R

2003-02-01

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function. Copyright 2003 Elsevier Science (USA)

Natural killer T cell facilitated engraftment of rat skin but not islet xenografts in mice.

PubMed

Gordon, Ethel J; Kelkar, Vinaya

2009-01-01

We have studied cellular components required for xenograft survival mediated by anti-CD154 monoclonal antibody (mAb) and a transfusion of donor spleen cells and found that the elimination of CD4(+) but not CD8(+) cells significantly improves graft survival. A contribution of other cellular components, such as natural killer (NK) cells and natural killer T (NKT) cells, for costimulation blockade-induced xenograft survival has not been clearly defined. We therefore tested the hypothesis that NK or NKT cells would promote rat islet and skin xenograft acceptance in mice. Lewis rat islets or skin was transplanted into wild type B6 mice or into B6 mice that were Jalpha18(null), CD1(null), or beta2 microglobulin (beta2M)(null) NK 1.1 depleted, or perforin(null). Graft recipients were pretreated with an infusion of donor derived spleen cells and a brief course of anti-CD154 mAb treatments. Additional groups received mAb or cells only. We first observed that the depletion of NK1.1 cells does not significantly interfere with graft survival in C57BL/6 (B6) mice. We used NKT cell deficient B6 mice to test the hypothesis that NKT cells are involved in islet and skin xenograft survival in our model. These mice bear a null mutation in the gene for the Jalpha18 component of the T-cell receptor. The component is uniquely associated with NKT cells. We found no difference in islet xenograft survival between Jalpha18(null) and wild type B6 mice. In contrast, median skin graft survival appeared shorter in Jalpha18(null) recipients. These data imply a role for Jalpha18(+) NKT cells in skin xenograft survival in treated mice. In order to confirm this inference, we tested skin xenograft survival in B6 CD1(null) mice because NKT cells are CD1 restricted. Results of these trials demonstrate that the absence of CD1(+) cells adversely affects rat skin graft survival. An additional assay in beta2M(null) mice demonstrated a requirement for major histocompatibility complex (MHC) class I expression in the graft host, and we demonstrate that CD1 is the requisite MHC component. We further demonstrated that, unlike reports for allograft survival, skin xenograft survival does not require perforin-secreting NK cells. We conclude that MHC class I(+) CD1(+) Jalpha18(+) NKT cells promote the survival of rat skin but not rat islet xenografts. These studies implicate different mechanisms for inducing and maintaining islet vs. skin xenograft survival in mice treated with donor antigen and anti-CD154 mAb, and further indicate a role for NKT cells but not NK cells in skin xenograft survival.

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3.

PubMed

Zhou, Gaofeng; Pereira, Jorge F; Delhaize, Emmanuel; Zhou, Meixue; Magalhaes, Jurandir V; Ryan, Peter R

2014-06-01

Malate and citrate efflux from root apices is a mechanism of Al(3+) tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al(3+)-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al(3+) tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al(3+)-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al(3+)-activated citrate efflux from root apices and greater tolerance to Al(3+) toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al(3+) tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al(3+) tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al(3+) tolerance of an important crop species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2013-09-01

accomplishments include creation of relevant plant lines, development of in vitro assays, and profiling of mRNA expression in null mutants. 15. SUBJECT TERMS...DNA methylation, UHRF1, VIM1, ubiquitination, epigenetics, chromatin 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF...Molecular Basis of Human Disease ,” which covered several weeks’ worth of material specifically related to the molecular and epigenetic basis of cancer

Polo-like kinase 1 is essential for early embryonic development and tumor suppression.

PubMed

Lu, Lin-Yu; Wood, Jamie L; Minter-Dykhouse, Katherine; Ye, Lin; Saunders, Thomas L; Yu, Xiaochun; Chen, Junjie

2008-11-01

Polo-like kinases (Plks) are serine/threonine kinases that are highly conserved in organisms from yeasts to humans. Previous reports have shown that Plk1 is critical for all stages of mitosis and may play a role in DNA replication during S phase. While much work has focused on Plk1, little is known about the physiological function of Plk1 in vivo. To address this question, we generated Plk1 knockout mice. Plk1 homozygous null mice were embryonic lethal, and early Plk1(-/-) embryos failed to survive after the eight-cell stage. Immunocytochemistry studies revealed that Plk1-null embryos were arrested outside the mitotic phase, suggesting that Plk1 is important for proper cell cycle progression. It has been postulated that Plk1 is a potential oncogene, due to its overexpression in a variety of tumors and tumor cell lines. While the Plk1 heterozygotes were healthy at birth, the incidence of tumors in these animals was threefold greater than that in their wild-type counterparts, demonstrating that the loss of one Plk1 allele accelerates tumor formation. Collectively, our data support that Plk1 is important for early embryonic development and may function as a haploinsufficient tumor suppressor.

The origin of nulls mode changes and timing noise in pulsars

NASA Astrophysics Data System (ADS)

Jones, P. B.

A solvable polar cap model obtained previously has normal states which may be associated with radio emission and null states. The solutions cannot be time-independent; the neutron star surface temperature T and mean surface nuclear charge Z are both functions of time. The normal and null states, and the transitions between them, form closed cycles in the T-Z plane. Normal-null transitions can occur inside a fraction of the area on the neutron star surface intersected by open magnetic flux lines. The fraction increases with pulsar period and becomes unity when the pulsar nears extinction. Frequency noise, mode changes, and pulse nulls have a common explanation in the transitions.

The origin of nulls, mode changes and timing noise in pulsars

NASA Astrophysics Data System (ADS)

Jones, P. B.

1982-09-01

A solvable polar cap model obtained previously has normal states which may be associated with radio emission, and null states. The solutions cannot be time-independent; the neutron star surface temperature T and mean surface nuclear charge Z are both functions of time. The normal and null states and the transitions between them, form closed cycles in the T-Z plane. Normal-null transitions can occur inside a fraction of the area of the neutron star surface intersected by open magnetic flux lines. The fraction increases with pulsar period and becomes unity when the pulsar nears extinction. Frequency noise, mode changes and pulse nulls have a common explanation in the transitions.

Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

NASA Technical Reports Server (NTRS)

Furlan, F.; Lecanda, F.; Screen, J.; Civitelli, R.

2001-01-01

Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

Stat1-independent regulation of gene expression in response to IFN-γ

PubMed Central

Ramana, Chilakamarti V.; Gil, M. Pilar; Han, Yulong; Ransohoff, Richard M.; Schreiber, Robert D.; Stark, George R.

2001-01-01

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling. PMID:11390994

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40

PubMed Central

McNees, Adrienne L.; Harrigal, Lindsay J.; Kelly, Aoife; Minard, Charles G.; Wong, Connie

2018-01-01

Background Polyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40–human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined. Results SV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant. Conclusion These findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells. Significance Lymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human lymphocytes. PMID:29432481

[Programmed mouse genome modifications].

PubMed

Babinet, C

1998-02-01

The availability, in the mouse, of embryonic stem cells (ES cells) which have the ability to colonize the germ line of a developing embryo, has opened entirely new avenues to the genetic approach of embryonic development, physiology and pathology of this animal. Indeed, it is now possible, using homologous recombination in ES cells, to introduce mutations in any gene as long as it has been cloned. Thus, null as well as more subtle mutations can be created. Furthermore, scenarios are currently being derived which will allow one to generate conditional mutations. Taken together, these methods offer a tremendous tool to study gene function in vivo; they also open the way to creating murine models of human genetic diseases.

Circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive T cells are independently associated with disease damage in systemic lupus erythematosus patients.

PubMed

Ugarte-Gil, M F; Sánchez-Zúñiga, C; Gamboa-Cárdenas, R V; Aliaga-Zamudio, M; Zevallos, F; Tineo-Pozo, G; Cucho-Venegas, J M; Mosqueira-Riveros, A; Medina, M; Perich-Campos, R A; Alfaro-Lozano, J L; Rodriguez-Bellido, Z; Alarcón, G S; Pastor-Asurza, C A

2016-03-01

To determine whether circulating CD4+CD28null and extra-thymic CD4+CD8+ double positive (DP) T cells are independently associated with damage accrual in systemic lupus erythematosus (SLE) patients. This cross-sectional study was conducted between September 2013 and April 2014 in consecutive SLE patients from our Rheumatology Department. CD4+CD28null and CD4+CD8+ DP T-cell frequencies were analyzed by flow-cytometry. The association of damage (SLICC/ACR Damage Index, SDI) and CD4+CD28null and CD4+CD8+ DP T cells was examined by univariable and multivariable Poisson regression models, adjusting for possible confounders. All analyses were performed using SPSS 21.0. Patients' (n = 133) mean (SD) age at diagnosis was 35.5 (16.8) years, 124 (93.2%) were female; all were mestizo (mixed Caucasian and Amerindian ancestry). Disease duration was 7.4 (6.8) years. The SLE Disease Activity Index was 5.5 (4.2), and the SDI 0.9 (1.2). The percentages of CD4+CD28null and CD4+CD8+ DP T cells were 17.1 (14.4) and 0.4 (1.4), respectively. The percentage of CD4+CD28null and CD4+CD8+ DP T cells were positively associated with a higher SDI in both univariable (rate ratio (RR) 1.02, 95% confidence interval (CI): 1.01-1.03 and 1.17, 95% CI: 1.07-1.27, respectively; p < 0.001 for both) and multivariable analyses RR 1.02, 95% CI: 1.01-1.03, p = 0.001 for CD4+CD28null T cells and 1.28, 95% CI: 1.13-1.44, p < 0.001 for CD4+CD8+ DP T cells). Only the renal domain remained associated with CD4+CD28null in multivariable analyses (RR 1.023 (1.002-1.045); p = 0.034). In SLE patients, CD4+CD28null and CD4+CD8+ DP T cells are independently associated with disease damage. Longitudinal studies are warranted to determine the predictive value of these associations. © The Author(s) 2015.

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation1[OPEN

PubMed Central

Bush, Maxwell S.; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M. K.; Mayberry, Laura K.; Browning, Karen S.

2016-01-01

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

A Gaussian Mixture Model for Nulling Pulsars

NASA Astrophysics Data System (ADS)

Kaplan, D. L.; Swiggum, J. K.; Fichtenbauer, T. D. J.; Vallisneri, M.

2018-03-01

The phenomenon of pulsar nulling—where pulsars occasionally turn off for one or more pulses—provides insight into pulsar-emission mechanisms and the processes by which pulsars turn off when they cross the “death line.” However, while ever more pulsars are found that exhibit nulling behavior, the statistical techniques used to measure nulling are biased, with limited utility and precision. In this paper, we introduce an improved algorithm, based on Gaussian mixture models, for measuring pulsar nulling behavior. We demonstrate this algorithm on a number of pulsars observed as part of a larger sample of nulling pulsars, and show that it performs considerably better than existing techniques, yielding better precision and no bias. We further validate our algorithm on simulated data. Our algorithm is widely applicable to a large number of pulsars even if they do not show obvious nulls. Moreover, it can be used to derive nulling probabilities of nulling for individual pulses, which can be used for in-depth studies.

Modular Hamiltonians on the null plane and the Markov property of the vacuum state

NASA Astrophysics Data System (ADS)

Casini, Horacio; Testé, Eduardo; Torroba, Gonzalo

2017-09-01

We compute the modular Hamiltonians of regions having the future horizon lying on a null plane. For a CFT this is equivalent to regions with a boundary of arbitrary shape lying on the null cone. These Hamiltonians have a local expression on the horizon formed by integrals of the stress tensor. We prove this result in two different ways, and show that the modular Hamiltonians of these regions form an infinite dimensional Lie algebra. The corresponding group of unitary transformations moves the fields on the null surface locally along the null generators with arbitrary null line dependent velocities, but act non-locally outside the null plane. We regain this result in greater generality using more abstract tools on the algebraic quantum field theory. Finally, we show that modular Hamiltonians on the null surface satisfy a Markov property that leads to the saturation of the strong sub-additive inequality for the entropies and to the strong super-additivity of the relative entropy.

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana.

PubMed

Tsutsui, Hiroki; Higashiyama, Tetsuya

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

PubMed

Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

2010-12-01

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

T cell-specific deletion of the inositol phosphatase SHIP reveals its role in regulating Th1/Th2 and cytotoxic responses

PubMed Central

Tarasenko, Tatyana; Kole, Hemanta K.; Chi, Anthony W.; Mentink-Kane, Margaret M.; Wynn, Thomas A.; Bolland, Silvia

2007-01-01

The 5′-phosphoinositol phosphatase SHIP negatively regulates signaling pathways triggered by antigen, cytokine and Fc receptors in both lymphocytes and myeloid cells. Mice with germ-line (null) deletion of SHIP develop a myeloproliferative-like syndrome that causes early lethality. Lymphocyte anomalies have been observed in SHIP-null mice, but it is unclear whether they are due to an intrinsic requirement of SHIP in these cells or a consequence of the severe myeloid pathology. To precisely address the function of SHIP in T cells, we have generated mice with T cell-specific deletion of SHIP. In the absence of SHIP, we found no differences in thymic selection or in the activation state and numbers of regulatory T cells in the periphery. In contrast, SHIP-deficient T cells do not skew efficiently to Th2 in vitro. Mice with T cell-specific deletion of SHIP show poor antibody responses on Alum/NP-CGG immunization and diminished Th2 cytokine production when challenged with Schistosoma mansoni eggs. The failure to skew to Th2 responses may be the consequence of increased basal levels of the Th1-associated transcriptional factor T-bet, resulting from enhanced sensitivity to cytokine-mediated T-bet induction. SHIP-deficient CD8+ cells show enhanced cytotoxic responses, consistent with elevated T-bet levels in these cells. Overall our experiments indicate that in T cells SHIP negatively regulates cytokine-mediated activation in a way that allows effective Th2 responses and limits T cell cytotoxicity. PMID:17585010

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

PubMed Central

Basu, Amitabha; Kligman, Lorraine H; Samulewicz, Stefan J; Howe, Chin C

2001-01-01

Background SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them. Results In large (25 mm) wounds, SPARC-null mice showed a significant delay in healing as compared to wild-type mice (31 days versus 24 days). Granulation tissue formation and extracellular matrix protein production were delayed in small 6 mm SPARC-null wounds initially but were resolved by day 6. In in vitro wound-healing assays, while wild-type primary dermal fibroblasts showed essentially complete wound closure at 11 hours, wound closure of SPARC-null cells was incomplete even at 31 hours. Addition of purified SPARC restored the normal time course of wound closure. Treatment of SPARC-null cells with mitomycin C to analyze cell migration without cell proliferation showed that wound repair remained incomplete after 31 hours. Cell proliferation as measured by 3H-thymidine incorporation and collagen gel contraction by SPARC-null cells were not compromised. Conclusions A significant delay in healing large excisional wounds and setback in granulation tissue formation and extracellular matrix protein production in small wounds establish that SPARC is required for granulation tissue formation during normal repair of skin wounds in mice. A defect in wound closure in vitro indicates that SPARC regulates cell migration. We conclude that SPARC plays a role in wound repair by promoting fibroblast migration and thus granulation tissue formation. PMID:11532190

Influence of pinches on magnetic reconnection in turbulent space plasmas

NASA Astrophysics Data System (ADS)

Olshevsky, Vyacheslav; Lapenta, Giovanni; Markidis, Stefano; Divin, Andrey

A generally accepted scenario of magnetic reconnection in space plasmas is the breakage of magnetic field lines in X-points. In laboratory, reconnection is widely studied in pinches, current channels embedded into twisted magnetic fields. No model of magnetic reconnection in space plasmas considers both null-points and pinches as peers. We have performed a particle-in-cell simulation of magnetic reconnection in a three-dimensional configuration where null-points are present nitially, and Z-pinches are formed during the simulation. The X-points are relatively stable, and no substantial energy dissipation is associated with them. On contrary, turbulent magnetic reconnection in the pinches causes the magnetic energy to decay at a rate of approximately 1.5 percent per ion gyro period. Current channels and twisted magnetic fields are ubiquitous in turbulent space plasmas, so pinches can be responsible for the observed high magnetic reconnection rates.

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

PubMed

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

CD4+CD28null T Cells are related to previous cytomegalovirus infection but not to accelerated atherosclerosis in ANCA-associated vasculitis.

PubMed

Slot, Marjan C; Kroon, Abraham A; Damoiseaux, Jan G M C; Theunissen, Ruud; Houben, Alfons J H M; de Leeuw, Peter W; Tervaert, Jan Willem Cohen

2017-05-01

Previous studies have suggested an increased risk for cardiovascular events in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). We analyzed the presence of atherosclerotic damage in patients with AAV in relation to the presence of CD4 + CD28 null T cells and antibodies against cytomegalovirus (CMV) and human Heat-Shock Protein 60 (hHSP60). In this cross-sectional study, patients with inactive AAV were compared with healthy controls (HC). Carotid intima-media thickness (IMT) and aortic pulse-wave velocity (PWV) were measured. In addition, CD4 + CD28 null T cells, anti-CMV, and anti-hHSP60 levels were determined. Forty patients with AAV were included. Patients' spouses were recruited as HC (N = 38). CD4 + CD28 null T cells are present in patients with AAV in a higher percentage (median 3.1, range 0.01-85) than in HC (0.28, 0-36, P < 0.0001). No significant difference in IMT (mm) between patients and controls was detected (mean 0.77 ± standard deviation 0.15 and 0.73 ± 0.11, respectively, P = 0.20). PWV standardized for MAP was increased in AAV patients (9.80 ± 2.50 m/s, compared to 8.72 ± 1.68 in HC, P = 0.04). There was a strong association between a previous CMV infection and the presence and percentage of CD4 + CD28 null T cells (0.33 vs 13.8, P < 0.001). There was no relationship between CD4 + CD28 null T cells and/or a previous CMV infection and IMT or PWV. There was no relation between anti-hHSP60 and CD4 + CD28 null T cells. Increased PWV values suggest atherosclerotic damage in patients with AAV. Plaque size, as determined by IMT, did not differ. CD4 + CD28 null T cells are increased in AAV and related to the previous CMV infection.

A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells.

PubMed

Kwiatkowski, David J; Zhang, Hongbing; Bandura, Jennifer L; Heiberger, Kristina M; Glogauer, Michael; el-Hashemite, Nisreen; Onda, Hiroaki

2002-03-01

Tuberous sclerosis (TSC) is a autosomal dominant genetic disorder caused by mutations in either TSC1 or TSC2, and characterized by benign hamartoma growth. We developed a murine model of Tsc1 disease by gene targeting. Tsc1 null embryos die at mid-gestation from a failure of liver development. Tsc1 heterozygotes develop kidney cystadenomas and liver hemangiomas at high frequency, but the incidence of kidney tumors is somewhat lower than in Tsc2 heterozygote mice. Liver hemangiomas were more common, more severe and caused higher mortality in female than in male Tsc1 heterozygotes. Tsc1 null embryo fibroblast lines have persistent phosphorylation of the p70S6K (S6K) and its substrate S6, that is sensitive to treatment with rapamycin, indicating constitutive activation of the mTOR-S6K pathway due to loss of the Tsc1 protein, hamartin. Hyperphosphorylation of S6 is also seen in kidney tumors in the heterozygote mice, suggesting that inhibition of this pathway may have benefit in control of TSC hamartomas.

Focal adhesion kinase is involved in mechanosensing during fibroblast migration

NASA Technical Reports Server (NTRS)

Wang, H. B.; Dembo, M.; Hanks, S. K.; Wang, Y.

2001-01-01

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

Prolyl 3-hydroxylase 1 and CRTAP are mutually stabilizing in the endoplasmic reticulum collagen prolyl 3-hydroxylation complex.

PubMed

Chang, Weizhong; Barnes, Aileen M; Cabral, Wayne A; Bodurtha, Joann N; Marini, Joan C

2010-01-15

Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta (OI) with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B (CyPB) in the endoplasmic reticulum (ER) which 3-hydroxylates the Pro986 residue of alpha1(I) and alpha1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. CyPB levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared with control cells and accounts for 15-20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of types VII and VIII OI.

Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression.

PubMed

Yuri, Shunsuke; Fujimura, Sayoko; Nimura, Keisuke; Takeda, Naoki; Toyooka, Yayoi; Fujimura, Yu-Ichi; Aburatani, Hiroyuki; Ura, Kiyoe; Koseki, Haruhiko; Niwa, Hitoshi; Nishinakamura, Ryuichi

2009-04-01

Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri-implantation and that Sall4-null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4-null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder-free Sall4-null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1-null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4-null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression.

Transforming Growth Factor Beta (TGFβ1, TGFβ2 and TGFβ3) Null-Mutant Phenotypes in Embryonic Gonadal Development

PubMed Central

Memon, Mushtaq A.; Anway, Matthew D.; Covert, Trevor R.; Uzumcu, Mehmet; Skinner, Michael K.

2008-01-01

The role transforming growth factor beta (TGFb) isoforms TGFb1, TGFb2 and TGFb3 have in the regulation of embryonic gonadal development was investigated with the use of null-mutant (i.e. knockout) mice for each of the TGFb isoforms. Late embryonic gonadal development was investigated because homozygote TGFb null-mutant mice generally die around birth, with some embryonic loss as well. In the testis, the TGFb1 null-mutant mice had a decrease in the number of germ cells at birth, postnatal day 0 (P0). In the testis, the TGFb2 null-mutant mice had a decrease in the number of seminiferous cords at embryonic day 15 (E15). In the ovary, the TGFb2 null-mutant mice had an increase in the number of germ cells at P0. TGFb isoforms appear to have a role in gonadal development, but interactions between the isoforms is speculated to compensate in the different TGFb isoform null-mutant mice. PMID:18790002

Experimental evaluation of achromatic phase shifters for mid-infrared starlight suppression.

PubMed

Gappinger, Robert O; Diaz, Rosemary T; Ksendzov, Alexander; Lawson, Peter R; Lay, Oliver P; Liewer, Kurt M; Loya, Frank M; Martin, Stefan R; Serabyn, Eugene; Wallace, James K

2009-02-10

Phase shifters are a key component of nulling interferometry, one of the potential routes to enabling the measurement of faint exoplanet spectra. Here, three different achromatic phase shifters are evaluated experimentally in the mid-infrared, where such nulling interferometers may someday operate. The methods evaluated include the use of dispersive glasses, a through-focus field inversion, and field reversals on reflection from antisymmetric flat-mirror periscopes. All three approaches yielded deep, broadband, mid-infrared nulls, but the deepest broadband nulls were obtained with the periscope architecture. In the periscope system, average null depths of 4x10(-5) were obtained with a 25% bandwidth, and 2x10(-5) with a 20% bandwidth, at a central wavelength of 9.5 mum. The best short term nulls at 20% bandwidth were approximately 9x10(-6), in line with error budget predictions and the limits of the current generation of hardware.

Intrathymic injection of hematopoietic progenitor cells establishes functional T cell development in a mouse model of severe combined immunodeficiency.

PubMed

Tuckett, Andrea Z; Thornton, Raymond H; O'Reilly, Richard J; van den Brink, Marcel R M; Zakrzewski, Johannes L

2017-05-16

Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice is feasible and facilitates the generation of functional T cells conferring protective immunity. Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase + , CD45.1 + ) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγ null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice was assessed in a B cell lymphoma model. Despite the small size of the thymic remnant in NOD-scid IL2rγ null mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγ null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγ null mice, suggesting that immune dysregulation is not a major concern. Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency.

Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice.

PubMed

Sferruzzi-Perri, Amanda N; Macpherson, Anne M; Roberts, Claire T; Robertson, Sarah A

2009-07-01

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

PubMed

Watson, Alan R; Pitchford, Simon C; Reynolds, Louise E; Direkze, Natalie; Brittan, Mairi; Alison, Malcolm R; Rankin, Sara; Wright, Nicholas A; Hodivala-Dilke, Kairbaan M

2010-03-01

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Loss of CD28 on Peripheral T Cells Decreases the Risk for Early Acute Rejection after Kidney Transplantation

PubMed Central

Dedeoglu, Burç; Meijers, Ruud W. J.; Klepper, Mariska; Hesselink, Dennis A.; Baan, Carla C.; Litjens, Nicolle H. R.; Betjes, Michiel G. H.

2016-01-01

Background End-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR). Methods 222 living donor kidney transplant recipients were prospectively analyzed. EAR was defined as biopsy proven acute allograft rejection within 3 months after kidney transplantation. The differentiation status of circulating T cells, the relative telomere length and the number of CD31+ naive T cells were determined as T-cell ageing parameters. Results Of the 222 patients analyzed, 30 (14%) developed an EAR. The donor age and the historical panel reactive antibody score were significantly higher (p = 0.024 and p = 0.039 respectively) and the number of related donor kidney transplantation was significantly lower (p = 0.018) in the EAR group. EAR-patients showed lower CD4+CD28null T-cell numbers (p<0.01) and the same trend was observed for CD8+CD28null T-cell numbers (p = 0.08). No differences regarding the other ageing parameters were found. A multivariate Cox regression analysis showed that higher CD4+CD28null T-cell numbers was associated with a lower risk for EAR (HR: 0.65, p = 0.028). In vitro, a significant lower percentage of alloreactive T cells was observed within CD28null T cells (p<0.001). Conclusion Immunological ageing-related expansion of highly differentiated CD28null T cells is associated with a lower risk for EAR. PMID:26950734

Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

PubMed Central

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

2013-01-01

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells

PubMed Central

Collins, Carol M.; Ellis, Joseph A.

2017-01-01

ABSTRACT Mutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo. PMID:28188262

Regulation of c–myc expression by IFN–γ through Stat1-dependent and -independent pathways

PubMed Central

Ramana, Chilakamarti V.; Grammatikakis, Nicholas; Chernov, Mikhail; Nguyen, Hannah; Goh, Kee Chuan; Williams, Bryan R.G.; Stark, George R.

2000-01-01

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c–myc expression. IFN–γ suppresses c–myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c–myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c–myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c–myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c–myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c–myc mRNA is induced, not suppressed, in response to IFN–γ. A role for Raf–1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf–1 complex. Both agents abrogated the IFN–γ-dependent induction of c–myc expression in Stat1-null cells. PMID:10637230

Inverse Relationship between Progesterone Receptor and Myc in Endometrial Cancer

PubMed Central

Dai, Donghai; Meng, Xiangbing; Thiel, Kristina W.; Leslie, Kimberly K.; Yang, Shujie

2016-01-01

Endometrial cancer, the most common gynecologic malignancy, is a hormonally-regulated disease. Response to progestin therapy positively correlates with hormone receptor expression, in particular progesterone receptor (PR). However, many advanced tumors lose PR expression. We recently reported that the efficacy of progestin therapy can be significantly enhanced by combining progestin with epigenetic modulators, which we term “molecularly enhanced progestin therapy.” What remained unclear was the mechanism of action and if estrogen receptor α (ERα), the principle inducer of PR, is necessary to restore functional expression of PR via molecularly enhanced progestin therapy. Therefore, we modeled advanced endometrial tumors that have lost both ERα and PR expression by generating ERα-null endometrial cancer cell lines. CRISPR-Cas9 technology was used to delete ERα at the genomic level. Our data demonstrate that treatment with a histone deacetylase inhibitor (HDACi) was sufficient to restore functional PR expression, even in cells devoid of ERα. Our studies also revealed that HDACi treatment results in marked downregulation of the oncogene Myc. We established that PR is a negative transcriptional regulator of Myc in endometrial cancer in the presence or absence of ERα, which is in contrast to studies in breast cancer cells. First, estrogen stimulation augmented PR expression and decreased Myc in endometrial cancer cell lines. Second, progesterone increased PR activity yet blunted Myc mRNA and protein expression. Finally, overexpression of PR by adenoviral transduction in ERα-null endometrial cancer cells significantly decreased expression of Myc and Myc-regulated genes. Analysis of the Cancer Genome Atlas (TCGA) database of endometrial tumors identified an inverse correlation between PR and Myc mRNA levels, with a corresponding inverse correlation between PR and Myc downstream transcriptional targets SRD5A1, CDK2 and CCNB1. Together, these data reveal a previously unanticipated inverse relationship between the tumor suppressor PR and the oncogene Myc in endometrial cancer. PMID:26859414

Neurogenin 1 Null Mutant Ears Develop Fewer, Morphologically Normal Hair Cells in Smaller Sensory Epithelia Devoid of Innervation

PubMed Central

Ma, Qiufu; Anderson, David J.

2000-01-01

The proneuronal gene neurogenin 1 (ngn1) is essential for development of the inner-ear sensory neurons that are completely absent in ngn1 null mutants. Neither afferent, efferent, nor autonomic nerve fibers were detected in the ears of ngn1 null mutants. We suggest that efferent and autonomic fibers are lost secondarily to the absence of afferents. In this article we show that ngn1 null mutants develop smaller sensory epithelia with morphologically normal hair cells. In particular, the saccule is reduced dramatically and forms only a small recess with few hair cells along a duct connecting the utricle with the cochlea. Hair cells of newborn ngn1 null mutants show no structural abnormalities, suggesting that embryonic development of hair cells is independent of innervation. However, the less regular pattern of dispersal within sensory epithelia may be caused by some effects of afferents or to the stunted growth of the sensory epithelia. Tracing of facial and stato-acoustic nerves in control and ngn1 null mutants showed that only the distal, epibranchial, placode-derived sensory neurons of the geniculate ganglion exist in mutants. Tracing further showed that these geniculate ganglion neurons project exclusively to the solitary tract. In addition to the normal complement of facial branchial and visceral motoneurons, ngn1 null mutants have some trigeminal motoneurons and contralateral inner-ear efferents projecting, at least temporarily, through the facial nerve. These data suggest that some neurons in the brainstem (e.g., inner-ear efferents, trigeminal motoneurons) require afferents to grow along and redirect to ectopic cranial nerve roots in the absence of their corresponding sensory roots. PMID:11545141

Transcriptional profiling reveals elevated Sox2 in DNA polymerase ß null mouse embryonic fibroblasts

PubMed Central

Li, Jianfeng; Luthra, Soumya; Wang, Xiao-Hong; Chandran, Uma R; Sobol, Robert W

2012-01-01

There are over 150 human proteins that have been categorized as bona fide DNA repair proteins. These DNA repair proteins maintain the integrity of the genome, reducing the onset of cancer, disease and aging phenotypes. Variations in expression and/or function would therefore impact genome integrity as well as the cellular response to genotoxins. Global gene expression analysis is an effective approach to uncover defects in DNA repair gene expression and to discover cellular and/or organismal effects brought about by external stimuli such as environmental genotoxicants, chemotherapeutic regimens, viral infections as well as developmental and age-related stimuli. Given the significance of genome stability in cell survival and response to stimuli, we have hypothesized that cells may undergo transcriptional re-programming to accommodate defects in basal DNA repair capacity to promote survival. As a test of this hypothesis, we have compared the transcriptome in three DNA polymerase ß knockout (Polß-KO) mouse embryonic fibroblasts (MEFs) and the corresponding wild-type (WT) littermate control cell lines. Each Polß-KO cell line was found to have a range of genes up-regulated, when compared to its WT littermate control cell line. Interestingly, six (6) genes were commonly up regulated in all three Polß-KO cell lines, including Sox2, one of several genes associated with the induction of pluripotent stem cells. Herein, we present these findings and suggest that loss of DNA repair and the induction of cellular transcriptional re-programming may, in part, contribute to tumor formation and the cellular response to external stimuli. PMID:23226616

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

PubMed

Pisconti, Addolorata; Banks, Glen B; Babaeijandaghi, Farshad; Betta, Nicole Dalla; Rossi, Fabio M V; Chamberlain, Jeffrey S; Olwin, Bradley B

2016-01-01

The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.

Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells

PubMed Central

Wu, Chun-Chi; Chen, Tzu-Hsiu; Liu, Bing-Lan; Wu, Li-Chen; Chen, Yung-Ching; Tzeng, Yew-Min; Hsu, Shih-Lan

2013-01-01

Destruxin B, isolated from entomopathogenic fungus Metarhizium anisopliae, is one of the cyclodepsipeptides with insecticidal and anticancer activities. In this study, destruxin B was extracted and purified by ion-exchange chromatography, silica gel chromatography, and semipreparative high-performance liquid chromatography. The potential anticancer effects and molecular mechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized. Our results showed that destruxin B induced apoptotic cell death in A549 cells. This event was accompanied by the activation of caspase-2, -3, and -9. Moreover, destruxin B increased the expression level of proapoptotic molecule, PUMA, while decreased antiapoptotic molecule Mcl-1. Additionally, the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatment. Knockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis in A549 cells. Interestingly, similar toxic effects and underlying mechanisms including caspase activation, upregulation of PUMA, and downregulation of Mcl-1 were also observed in a p53-null lung cancer H1299 cell line upon destruxin B treatment. Taken together, our findings suggest that destruxin-B-induced apoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway. PMID:24204395

Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

PubMed

Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

2014-07-01

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

PubMed Central

Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-01

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

PubMed

Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-19

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

Tropomodulin 1 Constrains Fiber Cell Geometry during Elongation and Maturation in the Lens Cortex

PubMed Central

Nowak, Roberta B.

2012-01-01

Lens fiber cells exhibit a high degree of hexagonal packing geometry, determined partly by tropomodulin 1 (Tmod1), which stabilizes the spectrin-actin network on lens fiber cell membranes. To ascertain whether Tmod1 is required during epithelial cell differentiation to fiber cells or during fiber cell elongation and maturation, the authors quantified the extent of fiber cell disorder in the Tmod1-null lens and determined locations of disorder by confocal microscopy and computational image analysis. First, nearest neighbor analysis of fiber cell geometry in Tmod1-null lenses showed that disorder is confined to focal patches. Second, differentiating epithelial cells at the equator aligned into ordered meridional rows in Tmod1-null lenses, with disordered patches first observed in elongating fiber cells. Third, as fiber cells were displaced inward in Tmod1-null lenses, total disordered area increased due to increased sizes (but not numbers) of individual disordered patches. The authors conclude that Tmod1 is required first to coordinate fiber cell shapes and interactions during tip migration and elongation and second to stabilize ordered fiber cell geometry during maturation in the lens cortex. An unstable spectrin-actin network without Tmod1 may result in imbalanced forces along membranes, leading to fiber cell rearrangements during elongation, followed by propagation of disorder as fiber cells mature. PMID:22473940

Amyloid precursor protein modulates ERK-1 and -2 signaling.

PubMed

Venezia, Valentina; Nizzari, Mario; Repetto, Emanuela; Violani, Elisabetta; Corsaro, Alessandro; Thellung, Stefano; Villa, Valentina; Carlo, Pia; Schettini, Gennaro; Florio, Tullio; Russo, Claudio

2006-12-01

The amyloid precursor protein (APP) is a transmembrane protein with a short cytoplasmic tail whose physiological function is unclear, although it is well documented that the proteolytic processing of APP could influence the development of Alzheimer's disease (AD) through the formation of membrane-bound C-terminal fragments (CTFs) and of beta-amyloid peptides (Abeta). We have recently shown that tyrosine-phosphorylated APP and CTFs may interact with Grb2 and ShcA adaptor proteins and that this coupling occurs at a higher extent in AD subjects only. To study the interaction between APP or CTFs and ShcA/Grb2 and to investigate their molecular target we have used as experimental model two different cell lines: H4 human neuroglioma cells and APP/APLP null mouse embryonic fibroblast cells (MEFs). Here we show that in H4 cells APP interacts with Grb2; conversely in APP/APLP-null MEF cells this interaction is possible only after the reintroduction of human APP by transfection. We have also shown that in MEF cells the transfection of a plasmid encoding for human APP wild-type enhances the phosphorylation of ERK-1 and -2 as revealed by Western blotting and immunofluorescence experiments. Finally, also in H4 cells the overexpression of APP upregulates the levels of phospho-ERK-1 and -2. In summary our data suggest that APP may influence phospho-ERK-1 and -2 signaling through its binding with Grb2 and ShcA adaptors. The meaning of this event is not clear, but APP interaction with these adaptors could be relevant to regulate mitogenic pathway.

Role of Tir and Intimin in the Virulence of Rabbit Enteropathogenic Escherichia coli Serotype O103:H2

PubMed Central

Marchès, Olivier; Nougayrède, Jean-Philippe; Boullier, Séverine; Mainil, Jacques; Charlier, Gérard; Raymond, Isabelle; Pohl, Pierre; Boury, Michèle; De Rycke, Jean; Milon, Alain; Oswald, Eric

2000-01-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity. PMID:10722617

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

PubMed

Marchès, O; Nougayrède, J P; Boullier, S; Mainil, J; Charlier, G; Raymond, I; Pohl, P; Boury, M; De Rycke, J; Milon, A; Oswald, E

2000-04-01

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Onecut1 and Onecut2 redundantly regulate early retinal cell fates during development

PubMed Central

Sapkota, Darshan; Chintala, Hemabindu; Wu, Fuguo; Fliesler, Steven J.; Hu, Zihua; Mu, Xiuqian

2014-01-01

Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general. PMID:25228773

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice.

PubMed

Fenske, Rachel J; Cadena, Mark T; Harenda, Quincy E; Wienkes, Haley N; Carbajal, Kathryn; Schaid, Michael D; Laundre, Erin; Brill, Allison L; Truchan, Nathan A; Brar, Harpreet; Wisinski, Jaclyn; Cai, Jinjin; Graham, Timothy E; Engin, Feyza; Kimple, Michelle E

2017-06-01

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes β-cell death and inhibits β-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional β-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive β-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive β-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, β-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a β-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in β-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of β-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response. Copyright © 2017 Endocrine Society.

Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression

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

Chen Wenshu; Yu Yichu; Lee Yijang

2010-06-01

Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin genemore » knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.« less

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

PubMed Central

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-01-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. PMID:23980694

The high-mobility-group box protein SSRP1/T160 is essential for cell viability in day 3.5 mouse embryos.

PubMed

Cao, Shang; Bendall, Heather; Hicks, Geoffrey G; Nashabi, Abudi; Sakano, Hitoshi; Shinkai, Yoichi; Gariglio, Marisa; Oltz, Eugene M; Ruley, H Earl

2003-08-01

The high-mobility-group (HMG) SSRP1 protein is a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, basal and regulated transcription, and DNA repair. To assist in the functional analysis of SSRP1, the Ssrp1 gene was targeted in murine embryonic stem cells, and the mutation was introduced into the germ line. Embryos homozygous for the targeted allele die soon after implantation, and preimplantation blastocysts are defective for cell outgrowth and/or survival in vitro. The Ssrp1 mutation was also crossed into a p53 null background without affecting growth and/or survival defects caused by loss of Ssrp1 function. Thus, Ssrp1 appears to encode nonredundant and p53-independent functions that are essential for cell viability.

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

Pancreatic Ductal Adenocarcinoma (PDA) mice lacking Mucin 1 have a profound defect in tumor growth and metastasis

PubMed Central

Besmer, Dahlia M.; Curry, Jennifer M.; Roy, Lopamudra D.; Tinder, Teresa L.; Sahraei, Mahnaz; Schettini, Jorge; Hwang, Sun-Il; Lee, Yong Y.; Gendler, Sandra J.; Mukherjee, Pinku

2011-01-01

MUC1 is over expressed and aberrantly glycosolated in >60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In the present study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared to both KC and KCM. Cell lines derived from KCKO tumors have significantly lower tumorigenic capacity compared to cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared to mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or matrix metalloproteinase-9 (MMP9). Further, significantly fewer KCKO cells entered the G2M phase of the cell cycle compared to the KCM cells. Proteomics and western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of MAPK, as well as a significant decrease in Nestin and Tubulin α-2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse in order to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro. PMID:21558393

Pancreatic ductal adenocarcinoma mice lacking mucin 1 have a profound defect in tumor growth and metastasis.

PubMed

Besmer, Dahlia M; Curry, Jennifer M; Roy, Lopamudra D; Tinder, Teresa L; Sahraei, Mahnaz; Schettini, Jorge; Hwang, Sun-Il; Lee, Yong Y; Gendler, Sandra J; Mukherjee, Pinku

2011-07-01

MUC1 is overexpressed and aberrantly glycosylated in more than 60% of pancreatic ductal adenocarcinomas. The functional role of MUC1 in pancreatic cancer has yet to be fully elucidated due to a dearth of appropriate models. In this study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1-null (KCKO) or express human MUC1 (KCM). We show that KCKO mice have significantly slower tumor progression and rates of secondary metastasis, compared with both KC and KCM. Cell lines derived from KCKO tumors have significantly less tumorigenic capacity compared with cells from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared with mice with KCM tumors. In vitro, KCKO cells have reduced proliferation and invasion and failed to respond to epidermal growth factor, platelet-derived growth factor, or matrix metalloproteinase 9. Further, significantly less KCKO cells entered the G(2)-M phase of the cell cycle compared with the KCM cells. Proteomics and Western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of mitogen-activated protein kinase (MAPK), as well as a significant decrease in nestin and tubulin-α2 chain expression in KCKO cells. Treatment with a MEK1/2 inhibitor, U0126, abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, suggesting that MUC1 is necessary for MAPK activity and oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro. ©2011 AACR

Targeted Metabolomics Reveals a Protective Role for Basal PPARα in Cholestasis Induced by α-Naphthylisothiocyanate.

PubMed

Dai, Manyun; Hua, Huiying; Lin, Hante; Xu, Gangming; Hu, Xiaowei; Li, Fei; Gonzalez, Frank J; Liu, Aiming; Yang, Julin

2018-04-06

α-Naphthylisothiocyanate (ANIT) is an experimental agent used to induce intrahepatic cholestasis. The Ppara-null mouse line is widely employed to explore the physiological and pathological roles of PPARα. However, little is known about how PPARα influences the hepatotoxicity of ANIT. In the present study, wild-type and Ppara-null mice were orally treated with ANIT to induce cholestasis. The serum metabolome of wild-type mice segregated from that of the Ppara-null mice, driven by changes of bile acid (BA) metabolites. Alkaline phosphatase and total BAs were elevated preferentially in Ppara-null mice, which correlated with changes in Cyp7a1, Cyp8b1, Mrp3, Cyp3a11, Cyp2b10, Ugt1a2, and Ugt1a5 genes and showed cross-talk between basal PPARα and potentially adaptive pathways. Il6, Tnfa, and target genes in the STAT3 pathway ( Socs3, Fga, Fgb, and Fgg) were up-regulated in Ppara-null mice but not in wild-type mice. The JNK pathway was activated in both mouse lines, while NF-κB and STAT3 were activated only in Ppara-null mice. These data suggest protection against cholestasis by basal PPARα involves regulation of BA metabolism and inhibition of NF-κB/STAT3 signaling. Considering studies on the protective effects of both basal and activated PPARα, caution should be exercised when one attempts to draw conclusions in which the PPARα is modified by genetic manipulation, fasting, or activation in pharmacological and toxicological studies.

Wnt-Responsive Cancer Stem Cells Are Located Close to Distorted Blood Vessels and Not in Hypoxic Regions in a p53-Null Mouse Model of Human Breast Cancer

PubMed Central

Landua, John D.; Bu, Wen; Wei, Wei; Li, Fuhai; Wong, Stephen T.C.; Dickinson, Mary E.; Rosen, Jeffrey M.; Lewis, Michael T.

2014-01-01

Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature. PMID:24797826

Wild-type cells rescue genotypically Math1-null hair cells in the inner ears of chimeric mice.

PubMed

Du, Xiaoping; Jensen, Patricia; Goldowitz, Daniel; Hamre, Kristin M

2007-05-15

The transcription factor Math1 has been shown to be critical in the formation of hair cells (HCs) in the inner ear. However, the influence of environmental factors in HC specification suggests that cell extrinsic factors are also crucial to their development. To test whether extrinsic factors impact development of Math1-null (Math1(beta-Gal/beta-Gal)) HCs, we examined neonatal (postnatal ages P0-P4.5) Math1-null chimeric mice in which genotypically mutant and wild-type cells intermingle to form the inner ear. We provide the first direct evidence that Math1-null HCs are able to be generated and survive in the conducive chimeric environment. beta-Galactosidase expression was used to identify genetically mutant cells while cells were phenotypically defined as HCs by morphological characteristics notably the expression of HC-specific markers. Genotypically mutant HCs were found in all sensory epithelia of the inner ear at all ages examined. Comparable results were obtained irrespective of the wild-type component of the chimeric mice. Thus, genotypically mutant cells retain the competence to differentiate into HCs. The implication is that the lack of the Math1 gene in HC precursors can be overcome by environmental influences, such as cell-cell interactions with wild-type cells, to ultimately result in the formation of HCs.

Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

PubMed

Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

2015-03-15

Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of starch synthase IIa gene dosage on grain, protein and starch in endosperm of wheat.

PubMed

Konik-Rose, Christine; Thistleton, Jenny; Chanvrier, Helene; Tan, Ihwa; Halley, Peter; Gidley, Michael; Kosar-Hashemi, Behjat; Wang, Hong; Larroque, Oscar; Ikea, Joseph; McMaugh, Steve; Regina, Ahmed; Rahman, Sadequr; Morell, Matthew; Li, Zhongyi

2007-11-01

Starch synthases (SS) are responsible for elongating the alpha-1,4 glucan chains of starch. A doubled haploid population was generated by crossing a line of wheat, which lacks functional ssIIa genes on each genome (abd), and an Australian wheat cultivar, Sunco, with wild type ssIIa alleles on each genome (ABD). Evidence has been presented previously indicating that the SGP-1 (starch granule protein-1) proteins present in the starch granule in wheat are products of the ssIIa genes. Analysis of 100 progeny lines demonstrated co-segregation of the ssIIa alleles from the three genomes with the SGP-1 proteins, providing further evidence that the SGP-1 proteins are the products of the ssIIa genes. From the progeny lines, 40 doubled haploid lines representing the eight possible genotypes for SSIIa (ABD, aBD, AbD, ABd, abD, aBd, Abd, abd) were characterized for their grain weight, protein content, total starch content and starch properties. For some properties (chain length distribution, pasting properties, swelling power, and gelatinization properties), a progressive change was observed across the four classes of genotypes (wild type, single nulls, double nulls and triple nulls). However, for other grain properties (seed weight and protein content) and starch properties (total starch content, granule morphology and crystallinity, granule size distribution, amylose content, amylose-lipid dissociation properties), a statistically significant change only occurred for the triple nulls, indicating that all three genes had to be missing or inactive for a change to occur. These results illustrate the importance of SSIIa in controlling grain and starch properties and the importance of amylopectin fine structure in controlling starch granule properties in wheat.

The impact of the SSIIa null mutations on grain traits and composition in durum wheat.

PubMed

Botticella, Ermelinda; Sestili, Francesco; Ferrazzano, Gianluca; Mantovani, Paola; Cammerata, Alessandro; D'Egidio, Maria Grazia; Lafiandra, Domenico

2016-09-01

Starch represents a major nutrient in the human diet providing essentially a source of energy. More recently the modification of its composition has been associated with new functionalities both at the nutritional and technological level. Targeting the major starch biosynthetic enzymes has been shown to be a valuable strategy to manipulate the amylose-amylopectin ratio in reserve starch. In the present work a breeding strategy aiming to produce a set of SSIIa (starch synthases IIa) null durum wheat is described. We have characterized major traits such as seed weight, total starch, amylose, protein and β-glucan content in a set of mutant families derived from the introgression of the SSIIa null trait into Svevo, an elite Italian durum wheat cultivar. A large degree of variability was detected and used to select wheat lines with either improved quality traits or agronomic performances. Semolina of a set of two SSIIa null lines showed new rheological behavior and an increased content of all major dietary fiber components, namely arabinoxylans, β-glucans and resistant starch. Furthermore the investigation of gene expression highlighted important differences in some genes involved in starch and β-glucans biosynthesis.

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

PubMed

Myer, A; Wagner, D S; Vivian, J L; Olson, E N; Klein, W H

1997-05-15

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells

PubMed Central

2014-01-01

Background PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. Results The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. PMID:24625059

Map LineUps: Effects of spatial structure on graphical inference.

PubMed

Beecham, Roger; Dykes, Jason; Meulemans, Wouter; Slingsby, Aidan; Turkay, Cagatay; Wood, Jo

2017-01-01

Fundamental to the effective use of visualization as an analytic and descriptive tool is the assurance that presenting data visually provides the capability of making inferences from what we see. This paper explores two related approaches to quantifying the confidence we may have in making visual inferences from mapped geospatial data. We adapt Wickham et al.'s 'Visual Line-up' method as a direct analogy with Null Hypothesis Significance Testing (NHST) and propose a new approach for generating more credible spatial null hypotheses. Rather than using as a spatial null hypothesis the unrealistic assumption of complete spatial randomness, we propose spatially autocorrelated simulations as alternative nulls. We conduct a set of crowdsourced experiments (n=361) to determine the just noticeable difference (JND) between pairs of choropleth maps of geographic units controlling for spatial autocorrelation (Moran's I statistic) and geometric configuration (variance in spatial unit area). Results indicate that people's abilities to perceive differences in spatial autocorrelation vary with baseline autocorrelation structure and the geometric configuration of geographic units. These results allow us, for the first time, to construct a visual equivalent of statistical power for geospatial data. Our JND results add to those provided in recent years by Klippel et al. (2011), Harrison et al. (2014) and Kay & Heer (2015) for correlation visualization. Importantly, they provide an empirical basis for an improved construction of visual line-ups for maps and the development of theory to inform geospatial tests of graphical inference.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

PubMed

Maier, Jennifer A; Harfe, Brian D

2011-11-15

The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF5-deficient mice

PubMed Central

Maier, Jennifer A.; Harfe, Brian D.

2011-01-01

Study Design The transition of the mouse embryonic notochord into nuclei pulposi was determined (“fate mapped”) in vivo in GDF-5 null mice using the Shhcre and R26R alleles. Objective To determine if abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5 null mice. Summary of Background Data The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5 null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5 null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or resulted from progressive postnatal degeneration of nuclei pulposi. Methods Gdf-5 mRNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5 null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24 week old mice. Results Our Gdf-5 mRNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate mapping experiments revealed that notochord cells in Gdf-5 null mice correctly form nuclei pulposi. Conclusion Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5 null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects. PMID:21278629

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.

PubMed

Zheng, Sisi; Zhou, Lijuan; Ma, Guolin; Zhang, Tian; Liu, Jindou; Li, Jia; Nguyen, Nhung T; Zhang, Xiaoyan; Li, Wanjie; Nwokonko, Robert; Zhou, Yandong; Zhao, Fukuan; Liu, Jingguo; Huang, Yun; Gill, Donald L; Wang, Youjun

2018-06-22

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1-491 and STIM1 1-666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

Branched-chain amino acids enhance cyst development in autosomal dominant polycystic kidney disease.

PubMed

Yamamoto, Junya; Nishio, Saori; Hattanda, Fumihiko; Nakazawa, Daigo; Kimura, Toru; Sata, Michio; Makita, Minoru; Ishikawa, Yasunobu; Atsumi, Tatsuya

2017-08-01

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney and liver cysts. The mammalian target of rapamycin (mTOR) cascade is one of the important pathways regulating cyst growth in ADPKD. Branched-chain amino acids (BCAAs), including leucine, play a crucial role to activate mTOR pathway. Therefore, we administered BCAA dissolved in the drinking water to Pkd1 flox/flox :Mx1-Cre (cystic) mice from four to 22 weeks of age after polyinosinic-polycytidylic acid-induced conditional Pkd1 knockout at two weeks of age. The BCAA group showed significantly greater kidney/body weight ratio and higher cystic index in both the kidney and liver compared to the placebo-treated mice. We found that the L-type amino acid transporter 1 that facilitates BCAA entry into cells is strongly expressed in cells lining the cysts. We also found increased cyst-lining cell proliferation and upregulation of mTOR and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways in the BCAA group. In vitro, we cultured renal epithelial cell lines from Pkd1 null mice with or without leucine. Leucine was found to stimulate cell proliferation, as well as activate mTOR and MAPK/ERK pathways in these cells. Thus, BCAA accelerated disease progression by mTOR and MAPK/ERK pathways. Hence, BCAA may be harmful to patients with ADPKD. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Shocks and currents in stratified atmospheres with a magnetic null point

NASA Astrophysics Data System (ADS)

Tarr, Lucas A.; Linton, Mark

2017-08-01

We use the resistive MHD code LARE (Arber et al 2001) to inject a compressive MHD wavepacket into a stratified atmosphere that has a single magnetic null point, as recently described in Tarr et al 2017. The 2.5D simulation represents a slice through a small ephemeral region or area of plage. The strong gradients in field strength and connectivity related to the presence of the null produce substantially different dynamics compared to the more slowly varying fields typically used in simple sunspot models. The wave-null interaction produces a fast mode shock that collapses the null into a current sheet and generates a set of outward propagating (from the null) slow mode shocks confined to field lines near each separatrix. A combination of oscillatory reconnection and shock dissipation ultimately raise the plasma's internal energy at the null and along each separatrix by 25-50% above the background. The resulting pressure gradients must be balanced by Lorentz forces, so that the final state has contact discontinuities along each separatrix and a persistent current at the null. The simulation demonstrates that fast and slow mode waves localize currents to the topologically important locations of the field, just as their Alfvenic counterparts do, and also illustrates the necessity of treating waves and reconnection as coupled phenomena.

Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.

PubMed

Jabbur, James R; Tabor, Amy D; Cheng, Xiaodong; Wang, Hua; Uesugi, Motonari; Lozano, Guillermina; Zhang, Wei

2002-10-10

Analyses of five wild-type p53 containing cell lines revealed lineage specific differences in phosphorylation of Thr18 after treatment with ionizing (IR) or ultraviolet (UV) radiation. Importantly, Thr18 phosphorylation correlated with induction of the p53 downstream targets p21(Waf1/Cip1) (p21) and Mdm-2, suggesting a transactivation enhancing role. Thr18 phosphorylation has been shown to abolish side-chain hydrogen bonding between Thr18 and Asp21, an interaction necessary for stabilizing alpha-helical conformation within the transactivation domain. Mutagenesis-derived hydrogen bond disruption attenuated the interaction of p53 with the transactivation repressor Mdm-2 but had no direct effect on the interaction of p53 with the basal transcription factor TAF(II)31. However, prior incubation of p53 mutants with Mdm-2 modulated TAF(II)31 interaction with p53, suggesting Mdm-2 blocks the accessibility of p53 to TAF(II)31. Consistently, p53-null cells transfected with hydrogen bond disrupting p53 mutants demonstrated enhanced endogenous p21 expression, whereas p53/Mdm-2-double null cells exhibited no discernible differences in p21 expression. We conclude disruption of intramolecular hydrogen bonding between Thr18 and Asp21 enhances p53 transactivation by modulating Mdm-2 binding, facilitating TAF(II)31 recruitment.

Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.

PubMed

Cao, Ou; Hoffman, Brad E; Moghimi, Babak; Nayak, Sushrusha; Cooper, Mario; Zhou, Shangzhen; Ertl, Hildegund C J; High, Katherine A; Herzog, Roland W

2009-10-01

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

PubMed

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C

2007-11-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

Msc1 Acts Through Histone H2A.Z to Promote Chromosome Stability in Schizosaccharomyces pombe

PubMed Central

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C.

2007-01-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage. PMID:17947424

INSL5 may be a unique marker of colorectal endocrine cells and neuroendocrine tumors

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

Mashima, Hirosato, E-mail: hmashima1-tky@umin.ac.jp; Ohno, Hideki; Yamada, Yumi

2013-03-22

Highlights: ► INSL5 is expressed in enteroendocrine cells along the colorectum. ► INSL5 is expressed increasingly from proximal colon to rectum. ► INSL5 co-localizes rarely with chromogranin A. ► All rectal neuroendocrine tumors examined expressed INSL5. -- Abstract: Insulin-like peptide 5 (INSL5) is a member of the insulin superfamily, and is a potent agonist for RXFP4. We have shown that INSL5 is expressed in enteroendocrine cells (EECs) along the colorectum with a gradient increase toward the rectum. RXFP4 is ubiquitously expressed along the digestive tract. INSL5-positive EECs have little immunoreactivity to chromogranin A (CgA) and might be a unique markermore » of colorectal EECs. CgA-positive EECs were distributed normally along the colorectum in INSL5 null mice, suggesting that INSL5 is not required for the development of CgA-positive EECs. Exogenous INSL5 did not affect the proliferation of human colon cancer cell lines, and chemically-induced colitis in INSL5 null mice did not show any significant changes in inflammation or mucosal healing compared to wild-type mice. In contrast, all of the rectal neuroendocrine tumors examined co-expressed INSL5 and RXFP4. INSL5 may be a unique marker of colorectal EECs, and INSL5–RXFP4 signaling might play a role in an autocrine/paracrine fashion in the colorectal epithelium and rectal neuroendocrine tumors.« less

Redundant role of tissue-selective TAF(II)105 in B lymphocytes.

PubMed

Freiman, Richard N; Albright, Shane R; Chu, Leslie E; Zheng, Shuang; Liang, Hong-Erh; Sha, William C; Tjian, Robert

2002-09-01

Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAF(II)s). The recent discovery of multiple TBP-related factors and tissue-specific TAF(II)s suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAF(II)105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAF(II)105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAF(II)105, we have generated TAF(II)105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAF(II)105. TAF(II)105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAF(II)105 in B cells is likely redundant with the function of other TAF(II)105-related cellular proteins.

Interchange Slip-Running Reconnection and Sweeping SEP-Beams

NASA Technical Reports Server (NTRS)

Masson, S.; Aulanier, G.; Pariat, E.; Klein, K.-L.

2011-01-01

We present a new model to explain how particles, accelerated at a reconnection site that is not magnetically connected to the Earth, could eventually propagate along the well-connected open flux tube. Our model is based on the results of a low-beta resistive magnetohydrodynamics simulation of a three-dimensional line-tied and initially current-free bipole, that is embedded in a non-uniform open potential field. The topology of this configuration is that of an asymmetric coronal null-point, with a closed fan surface and an open outer spine. When driven by slow photospheric shearing motions, field lines, initially fully anchored below the fan dome, reconnect at the null point, and jump to the open magnetic domain. This is the standard interchange mode as sketched and calculated in 2D. The key result in 3D is that, reconnected open field lines located in the vicinity of the outer spine, keep reconnecting continuously, across an open quasi-separatrix layer, as previously identified for non-open-null-point reconnection. The apparent slipping motion of these field lines leads to form an extended narrow magnetic flux tube at high altitude. Because of the slip-running reconnection, we conjecture that if energetic particles would be travelling through, or be accelerated inside, the diffusion region, they would be successively injected along continuously reconnecting field lines that are connected farther and farther from the spine. At the scale of the full Sun, owing to the super-radial expansion of field lines below 3 solar radius, such energetic particles could easily be injected in field lines slipping over significant distances, and could eventually reach the distant flux tube that is well-connected to the Earth.

The Simple Map for a Single-null Divertor Tokamak: How to Find the Footprint of Field lines

NASA Astrophysics Data System (ADS)

Figgins, Montoya; Ali, Halima; Punjabi, Alkesh

2000-10-01

We are working with the Simple Map^1 to find the footprint of field lines on the diverter plate in a single-null tokamak. Footprint of a field line is the position of the line when it escapes across the divertor plate. The Simple Map represents the magnetic field in a single-null divertor tokamak. The path of a field line is given by the equations: X_n+1=X_n-kY_n(1-Y_n) and Y_n+1=Y_n+kX_n+1. In order to find the footprint, we must first find the last good surface which is Y=0.997135768 and X=0. The value of k is fixed at 0.6. The starting values X0 are fixed at X_0=0. We use 10,000 points between the last good surface and the X-point. The X-point is located at (0,1). We also use the Continuous Analog of the Simple Map given by the equations: X(φ)=X_0-kY0 (1-Y_0)φ and Y(φ)=Y_0+kX(φ)φ. This will tell us what the (φ,X) is which represents the field lines crossing the divertor plate. The divertor plate is located at Y=1. When graphed, the footprint of field lines looks like the rings of Saturn. This work is supported by US DOES OFES. Ms. Montoya Figgins is HU CFRT Summer Fusion High School Scholar from E. E. Smith High School in North Carolina. She is supported by NASA under its NASA SHARP Plus Program. 1. Punjabi A, Verma A, and Boozer A, Phys Rev Lett, 69, 3322 (1992) and J Plasma Phys, 52, 91 (1994)

Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications

PubMed Central

Lange, Sara E.S.; Zheleznyak, Alex; Studer, Matthew; O'Shannessy, Daniel J.; Lapi, Suzanne E.; Van Tine, Brian A.

2016-01-01

Purpose The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab. Results Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs. Experimental Design Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed. Conclusion 89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy. PMID:26909615

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease

PubMed Central

Westbroek, Wendy; Nguyen, Matthew; Siebert, Marina; Lindstrom, Taylor; Burnett, Robert A.; Aflaki, Elma; Jung, Olive; Tamargo, Rafael; Rodriguez-Gil, Jorge L.; Acosta, Walter; Hendrix, An; Behre, Bahafta; Tayebi, Nahid; Fujiwara, Hideji; Sidhu, Rohini; Renvoise, Benoit; Ginns, Edward I.; Dutra, Amalia; Pak, Evgenia; Cramer, Carole; Ory, Daniel S.; Pavan, William J.

2016-01-01

ABSTRACT Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1. Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1. To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba−/− mice and the control littermate (gba+/+) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba−/− neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba+/+ neurons. This null allele gba−/− mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies. PMID:27482815

Regulation of the proliferation of colon cancer cells by compounds that affect glycolysis, including 3-bromopyruvate, 2-deoxyglucose and biguanides.

PubMed

Lea, Michael A; Qureshi, Mehreen S; Buxhoeveden, Michael; Gengel, Nicolette; Kleinschmit, Jessica; Desbordes, Charles

2013-02-01

In previous studies performed by our group, we observed that 2-deoxyglucose blocked the acidification of the medium used for culture of colon cancer cells caused by incubation with biguanides and it had an additive inhibitory effect on growth. In the present work, we found that 3-bromopyruvate can also prevent the lowering of pH caused by biguanide treatment. 3-Bromopyruvate inhibited colonic cancer cell proliferation, but the effect was not always additive to that of biguanides and an additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation of wild-type and p53-null cells and affected colonic cancer lines with different growth rates.

Regulation of the Proliferation of Colon Cancer Cells by Compounds that Affect Glycolysis, Including 3-Bromopyruvate, 2-Deoxyglucose and Biguanides

PubMed Central

Lea, Michael A.; Qureshi, Mehreen S.; Buxhoeveden, Michael; Gengel, Nicolette; Kleinschmit, Jessica; desBordes, Charles

2013-01-01

In previous studies we observed that 2-deoxyglucose blocked the acidification of the medium used for culture of colon cancer cells caused by incubation with biguanides and had an additive inhibitory effect on growth. In the present work, we found that 3-bromopyruvate can also prevent the lowering of pH caused by biguanide treatment. 3-Bromopyruvate inhibited colonic cancer cell proliferation but the effect was not always additive to that of biguanides and an additive effect was more notable in combined treatment with 3-bromopyruvate and 2-deoxyglucose. The induction of alkaline phosphatase activity by butyrate was not consistently affected by combination with other agents that modified glucose metabolism. The drug combinations that were examined inhibited proliferation of wild-type and P53 null cells and affected colonic cancer lines with different growth rates. PMID:23393330

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

PubMed

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

Nuclear Receptor TLX Regulates Cell Cycle Progression in Neural Stem Cells of the Developing Brain

PubMed Central

Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

2008-01-01

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain. PMID:17901127

Two-Wire to Four-Wire Audio Converter

NASA Technical Reports Server (NTRS)

Talley, G. L., Jr; Seale, B. L.

1983-01-01

Simple circuit provides interface between normally incompatible voicecommunication lines. Circuit maintains 40 dB of isolation between input and output halves of four-wire line permitting two-wire line to be connected. Balancing potentiometer, Rg, adjusts gain of IC2 to null feed through from input to output. Adjustment is done on workbench just after assembly.

Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

NASA Technical Reports Server (NTRS)

Fritzsch, B.; Signore, M.; Simeone, A.

2001-01-01

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.

Are polymorphisms in metabolism protective or a risk for reduced white blood cell counts in a Chinese population with low occupational benzene exposures?

PubMed Central

Ye, Ling-li; Zhang, Guang-hui; Huang, Jing-wen; Li, Yong; Zheng, Guo-qiao; Zhang, De-ting; Zhou, Li-fang; Tao, Xi-dan; Zhang, Jing; Ye, Yun-jie; Sun, Pin; Frank, Arthur; Xia, Zhao-lin

2015-01-01

Background: Genetic variations in metabolic enzyme genes may enhance hematotoxicity in benzene-exposed populations. Objective: To investigate the association between polymorphisms of metabolism genes and white blood cells (WBCs). Methods: Three hundred and eighty-five benzene-exposed workers and 220 unexposed indoor workers were recruited in China. We explored the relationship between metabolic enzymes polymorphisms [glutathione S-transferase T1/M1 (GSTT1/M1) null, glutathione S-transferase P1 (GSTP1)rs1695, Cytochrome P450 2E1 (CYP2E1) rs3813867, rs2031920, rs6413432, microsomal epoxide hydrolase (mEH) rs1051740, rs2234922] by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) analysis and WBC. Results: The exposed group had lower WBC counts (P<0.001) than the unexposed group. Increased susceptibility to hematotoxicity, as evidenced by lower WBC counts, was found in workers with null-GSTT1 (P = 0.045), null-GSTM1 (P = 0.030), rs2031920 (P = 0.020), and rs3813867 (P = 0.014) genotypes. White blood cell counts were also lower in workers with null-GSTT1 and null-GSTM after adjusting for age, gender, smoking, and alcohol consumption. Conclusion: Null-GSTT1 and null-GSTM1 genotypes and Cytochrome P4502E1 (CYP2E1: rs2031920, rs3813867) may support the hematotoxicity of benzene-exposed workers in China, and we can make use of it to select susceptible population. PMID:26179485

Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture: a model system to understand cancer cell growth mechanics.

PubMed

Pomo, Joseph M; Taylor, Robert M; Gullapalli, Rama R

2016-01-01

Spheroid based culture methods are gaining prominence to elucidate the role of the microenvironment in liver carcinogenesis. Additionally, the phenomenon of epithelial-mesenchymal transition also plays an important role in determining the metastatic potential of liver cancer. Tumor spheroids are thus important models to understand the basic biology of liver cancer. We cultured, characterized and examined the formation of compact 3-D micro-tumor spheroids in five hepatocellular carcinoma (HCC) cell lines, each with differing TP53 mutational status (wt vs mutant vs null). Spheroid viability and death was systematically measured over a course of a 10 day growth period using various assays. We also examined the TP53 and E-cadherin (CDH1) mRNA and protein expression status in each cell line of the 2-D and 3-D cell models. A novel finding of our study was the identification of variable 3-D spheroid morphology in individual cell lines, ranging from large and compact, to small and unstable spheroid morphologies. The observed morphological differences between the spheroids were robust and consistent over the duration of spheroid culture growth of 10 days in a repeatable manner. Highly variable CDH1 expression was identified depending on the TP53 mutational status of the individual HCC cell line, which may explain the variable spheroid morphology. We observed consistent patterns of TP53 and CDH1 expression in both 2-D and 3-D culture models. In conclusion, we show that 3-D spheroids are a useful model to determine the morphological growth characteristics of cell lines which are not immediately apparent in routine 2-D culture methods. 3-D culture methods may provide a better alternative to study the process of epithelial-mesenchymal transition (EMT) which is important in the process of liver cancer metastasis.

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

PubMed

Okuwa, T; Katayama, T; Takano, A; Kodaira, K; Yasukawa, H

2001-12-01

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes.

PubMed

Hodge, Greg; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2015-01-09

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR). Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy. COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = -.615; ) and with FEV1 in COPD (R = -.777). COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.

Impaired proteoglycan glycosylation, elevated TGF-β signaling, and abnormal osteoblast differentiation as the basis for bone fragility in a mouse model for gerodermia osteodysplastica

PubMed Central

Chan, Wing Lee; Steiner, Magdalena; Egerer, Johannes; Mizumoto, Shuji; Pestka, Jan M.; Zhang, Haikuo; Khayal, Layal Abo; Ott, Claus-Eric; Kolanczyk, Mateusz; Schinke, Thorsten; Paganini, Chiara; Rossi, Antonio; Sugahara, Kazuyuki; Amling, Michael; Knaus, Petra; Chan, Danny; Mundlos, Stefan

2018-01-01

Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment. PMID:29561836

Hypothesis Testing Using Spatially Dependent Heavy Tailed Multisensor Data

DTIC Science & Technology

2014-12-01

Office of Research 113 Bowne Hall Syracuse, NY 13244 -1200 ABSTRACT HYPOTHESIS TESTING USING SPATIALLY DEPENDENT HEAVY-TAILED MULTISENSOR DATA Report...consistent with the null hypothesis of linearity and can be used to estimate the distribution of a test statistic that can discrimi- nate between the null... Test for nonlinearity. Histogram is generated using the surrogate data. The statistic of the original time series is represented by the solid line

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth.

PubMed

Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H; Galipeau, Jacques; Deng, Xingming

2016-05-10

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth

PubMed Central

Deng, Jiusheng; Park, Dongkyoo; Wang, Mengchang; Nooka, Ajay; Deng, Qiaoya; Matulis, Shannon; Kaufman, Jonathan; Lonial, Sagar; Boise, Lawrence H.; Galipeau, Jacques; Deng, Xingming

2016-01-01

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy. PMID:27049723

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

The Landscape of Somatic Chromosomal Copy Number Aberrations in GEM Models of Prostate Carcinoma

PubMed Central

Bianchi-Frias, Daniella; Hernandez, Susana A.; Coleman, Roger; Wu, Hong; Nelson, Peter S.

2015-01-01

Human prostate cancer (PCa) is known to harbor recurrent genomic aberrations consisting of chromosomal losses, gains, rearrangements and mutations that involve oncogenes and tumor suppressors. Genetically engineered mouse (GEM) models have been constructed to assess the causal role of these putative oncogenic events and provide molecular insight into disease pathogenesis. While GEM models generally initiate neoplasia by manipulating a single gene, expression profiles of GEM tumors typically comprise hundreds of transcript alterations. It is unclear whether these transcriptional changes represent the pleiotropic effects of single oncogenes, and/or cooperating genomic or epigenomic events. Therefore, it was determined if structural chromosomal alterations occur in GEM models of PCa and whether the changes are concordant with human carcinomas. Whole genome array-based comparative genomic hybridization (CGH) was used to identify somatic chromosomal copy number aberrations (SCNAs) in the widely used TRAMP, Hi-Myc, Pten-null and LADY GEM models. Interestingly, very few SCNAs were identified and the genomic architecture of Hi-Myc, Pten-null and LADY tumors were essentially identical to the germline. TRAMP neuroendocrine carcinomas contained SCNAs, which comprised three recurrent aberrations including a single copy loss of chromosome 19 (encoding Pten). In contrast, cell lines derived from the TRAMP, Hi-Myc, and Pten-null tumors were notable for numerous SCNAs that included copy gains of chromosome 15 (encoding Myc) and losses of chromosome 11 (encoding p53). PMID:25298407

Divergence of IL-1, IL-18, and cell death in NLRP3 inflammasomopathies

PubMed Central

Brydges, Susannah D.; Broderick, Lori; McGeough, Matthew D.; Pena, Carla A.; Mueller, James L.; Hoffman, Hal M.

2013-01-01

The inflammasome is a cytoplasmic multiprotein complex that promotes proinflammatory cytokine maturation in response to host- and pathogen-derived signals. Missense mutations in cryopyrin (NLRP3) result in a hyperactive inflammasome that drives overproduction of the proinflammatory cytokines IL-1β and IL-18, leading to the cryopyrin-associated periodic syndromes (CAPS) disease spectrum. Mouse lines harboring CAPS-associated mutations in Nlrp3 have elevated levels of IL-1β and IL-18 and closely mimic human disease. To examine the role of inflammasome-driven IL-18 in murine CAPS, we bred Nlrp3 mutations onto an Il18r-null background. Deletion of Il18r resulted in partial phenotypic rescue that abolished skin and visceral disease in young mice and normalized serum cytokines to a greater extent than breeding to Il1r-null mice. Significant systemic inflammation developed in aging Nlrp3 mutant Il18r-null mice, indicating that IL-1 and IL-18 drive pathology at different stages of the disease process. Ongoing inflammation in double-cytokine knockout CAPS mice implicated a role for caspase-1–mediated pyroptosis and confirmed that CAPS is inflammasome dependent. Our results have important implications for patients with CAPS and residual disease, emphasizing the need to explore other NLRP3-mediated pathways and the potential for inflammasome-targeted therapy. PMID:24084736

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

PubMed

Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

2013-10-01

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. © 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High-Throughput Cell-Based Screens

DTIC Science & Technology

2005-02-01

Akt in the P13K pathway. Given the emerging data for a positive feedback loop induced by mTOR inhibition, a bispecific 5 inhibitor might be attractive...cells relatively sensitive to rapamycin are also sensitive to thioridazine. PTEN null cells are known to be preferentially sensitized to mTOR ...a potent mTOR inhibitor, a downstream protein kinase in the Akt pathway. Rapamycin showed strong growth inhibitory effect in PTEN-null cells but 786

Targeting the 5T4 oncofetal glycoprotein with an antibody drug conjugate (A1mcMMAF) improves survival in patient-derived xenograft models of acute lymphoblastic leukemia.

PubMed

McGinn, Owen J; Krishnan, Shekhar; Bourquin, Jean-Pierre; Sapra, Puja; Dempsey, Clare; Saha, Vaskar; Stern, Peter L

2017-06-01

Outcome in childhood acute lymphoblastic leukemia is prognosticated from levels of minimal residual disease after remission induction therapy. Higher levels of minimal residual disease are associated with inferior results even with intensification of therapy, thus suggesting that identification and targeting of minimal residual disease cells could be a therapeutic strategy. Here we identify high expression of 5T4 in subclonal populations of patient-derived xenografts from patients with high, post-induction levels of minimal residual disease. 5T4-positive cells showed preferential ability to overcome the NOD- scid IL2Rγ null mouse xenograft barrier, migrated in vitro on a CXCL12 gradient, preferentially localized to bone marrow in vivo and displayed the ability to reconstitute the original clonal composition on limited dilution engraftment. Treatment with A1mcMMAF (a 5T4-antibody drug conjugate) significantly improved survival without overt toxicity in mice engrafted with a 5T4-positive acute lymphoblastic leukemia cell line. Mice engrafted with 5T4-positive patient-derived xenograft cells were treated with combination chemotherapy or dexamethasone alone and then given A1mcMMAF in the minimal residual disease setting. Combination chemotherapy was toxic to NOD- scid IL2Rγ null mice. While dexamethasone or A1mcMMAF alone improved outcomes, the sequential administration of dexamethasone and A1mcMMAF significantly improved survival ( P =0.0006) over either monotherapy. These data show that specifically targeting minimal residual disease cells improved outcomes and support further investigation of A1mcMMAF in patients with high-risk B-cell precursor acute lymphoblastic leukemia identified by 5T4 expression at diagnosis. Copyright© Ferrata Storti Foundation.

Targeting the 5T4 oncofetal glycoprotein with an antibody drug conjugate (A1mcMMAF) improves survival in patient-derived xenograft models of acute lymphoblastic leukemia

PubMed Central

McGinn, Owen J.; Krishnan, Shekhar; Bourquin, Jean-Pierre; Sapra, Puja; Dempsey, Clare; Saha, Vaskar; Stern, Peter L.

2017-01-01

Outcome in childhood acute lymphoblastic leukemia is prognosticated from levels of minimal residual disease after remission induction therapy. Higher levels of minimal residual disease are associated with inferior results even with intensification of therapy, thus suggesting that identification and targeting of minimal residual disease cells could be a therapeutic strategy. Here we identify high expression of 5T4 in subclonal populations of patient-derived xenografts from patients with high, post-induction levels of minimal residual disease. 5T4-positive cells showed preferential ability to overcome the NOD-scidIL2Rγnull mouse xenograft barrier, migrated in vitro on a CXCL12 gradient, preferentially localized to bone marrow in vivo and displayed the ability to reconstitute the original clonal composition on limited dilution engraftment. Treatment with A1mcMMAF (a 5T4-antibody drug conjugate) significantly improved survival without overt toxicity in mice engrafted with a 5T4-positive acute lymphoblastic leukemia cell line. Mice engrafted with 5T4-positive patient-derived xenograft cells were treated with combination chemotherapy or dexamethasone alone and then given A1mcMMAF in the minimal residual disease setting. Combination chemotherapy was toxic to NOD-scidIL2Rγnull mice. While dexamethasone or A1mcMMAF alone improved outcomes, the sequential administration of dexamethasone and A1mcMMAF significantly improved survival (P=0.0006) over either monotherapy. These data show that specifically targeting minimal residual disease cells improved outcomes and support further investigation of A1mcMMAF in patients with high-risk B-cell precursor acute lymphoblastic leukemia identified by 5T4 expression at diagnosis. PMID:28341731

RNA-guided transcriptional regulation

DOEpatents

Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

2016-02-23

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

A rapid quantification of binocular misalignment without recording eye movements: Vertical and torsional alignment nulling.

PubMed

Beaton, Kara H; Shelhamer, Mark J; Roberts, Dale C; Schubert, Michael C

2017-05-01

Small, innate asymmetries between the left and right otolith organs can cause ocular misalignment with symptoms that include double vision and motion sickness. Additionally, ocular misalignment affects nearly 5% of the US population. We have developed a portable, non-invasive technology that uses subjective perception of binocular visual signals to estimate relative binocular alignment. The Vertical Alignment Nulling (VAN) and Torsional Alignment Nulling (TAN) tests ask subjects to view one red and one blue line on a tablet computer while looking through color-matched red and blue filters so that each eye sees only one of the lines. Subjects align the red and blue lines, which are initially vertically offset from one another during VAN or rotated relative to one another during TAN, until they perceive a single continuous line. Ocular misalignments are inferred from actual offsets in the final line positions. During testing, all binocular visual cues are eliminated by employing active-matrix organic light-emitting diode (AMOLED) technology and testing in darkness. VAN and TAN can accurately account for visual offsets induced by prisms, and test-retest reliability is excellent, with resolution better than many current standard clinical tests. VAN and TAN tests are similar to the clinical Lancaster red-green test. However, VAN and TAN employ inexpensive, hand-held hardware that can be self-administered with results that are quickly quantifiable. VAN and TAN provide simple, sensitive, and quantitative measures of binocular positioning alignment that may be useful for detecting subtle abnormalities in ocular positioning. Copyright © 2017 Elsevier B.V. All rights reserved.

Xenogeneic graft-versus-host-disease in NOD-scid IL-2Rγnull mice display a T-effector memory phenotype.

PubMed

Ali, Niwa; Flutter, Barry; Sanchez Rodriguez, Robert; Sharif-Paghaleh, Ehsan; Barber, Linda D; Lombardi, Giovanna; Nestle, Frank O

2012-01-01

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.

Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγnull Mice Display a T-Effector Memory Phenotype

PubMed Central

Ali, Niwa; Flutter, Barry; Sanchez Rodriguez, Robert; Sharif-Paghaleh, Ehsan; Barber, Linda D.; Lombardi, Giovanna; Nestle, Frank O.

2012-01-01

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; “Hu-PBMC mice”) are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγnull), notably the NOD-scid IL-2Rγnull (NSG) and BALB/c-Rag2 null IL-2Rγnull (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45+ compartment and higher engraftment levels of CD3+ T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (TEM) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting TEM-cell driven GvHD. PMID:22937164

Loss of miR-223 and JNK Signaling Contribute to Elevated Stathmin in Malignant Pleural Mesothelioma.

PubMed

Birnie, Kimberly A; Yip, Yan Y; Ng, Dominic C H; Kirschner, Michaela B; Reid, Glen; Prêle, Cecilia M; Musk, Arthur W Bill; Lee, Y C Gary; Thompson, Philip J; Mutsaers, Steven E; Badrian, Bahareh

2015-07-01

Malignant pleural mesothelioma (MPM) is often fatal, and studies have revealed that aberrant miRNAs contribute to MPM development and aggressiveness. Here, a screen of miRNAs identified reduced levels of miR-223 in MPM patient specimens. Interestingly, miR-223 targets Stathmin (STMN1), a microtubule regulator that has been associated with MPM. However, whether miR-223 regulates STMN1 in MPM and the functions of miR-223 and STMN1 in this disease are yet to be determined. STMN1 is also regulated by c-Jun N-terminal kinase (JNK) signaling, but whether this occurs in MPM and whether miR-223 plays a role are unknown. The relationship between STMN1, miR-223, and JNK was assessed using MPM cell lines, cells from pleural effusions, and MPM tissue. Evidence indicates that miR-223 is decreased in all MPM tissue compared with normal/healthy tissue. Conversely, STMN1 expression was higher in MPM cell lines when compared with primary mesothelial cell controls. Following overexpression of miR-223 in MPM cell lines, STMN1 levels were reduced, cell motility was inhibited, and tubulin acetylation induced. Knockdown of STMN1 using siRNAs led to inhibition of MPM cell proliferation and motility. Finally, miR-223 levels increased while STMN1 was reduced following the re-expression of the JNK isoforms in JNK-null murine embryonic fibroblasts, and STMN1 was reduced in MPM cell lines following the activation of JNK signaling. miR-223 regulates STMN1 in MPM, and both are in turn regulated by the JNK signaling pathway. As such, miR-223 and STMN1 play an important role in regulating MPM cell motility and may be therapeutic targets. ©2015 American Association for Cancer Research.

CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFκB signaling pathways

PubMed Central

Wang, Chaoqun; Fok, Kin Lam; Cai, Zhiming; Chen, Hao; Chan, Hsiao Chang

2017-01-01

CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway. PMID:27902973

CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFκB signaling pathways.

PubMed

Wang, Chaoqun; Fok, Kin Lam; Cai, Zhiming; Chen, Hao; Chan, Hsiao Chang

2017-01-10

CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway.

Yeast Genes Controlling Responses to Topogenic Signals in a Model Transmembrane Protein

PubMed Central

Tipper, Donald J.; Harley, Carol A

2002-01-01

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2–3% by a strong charge difference signal. Identified pio mutants increased this to 9–12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca2+ ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins. PMID:11950929

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

Noda, Kazuo, E-mail: knoda@kuhp.kyoto-u.ac.jp; Nakamura, Tomoyuki; Komatsu, Yoshihiro

Craniofacial sutures govern the shape of the craniofacial skeleton during postnatal development. The differentiation of suture mesenchymal cells to osteoblasts is precisely regulated in part by signaling through cell surface receptors that interact with extracellular proteins. Here we report that fibulin-5, a key extracellular matrix protein, is important for craniofacial skeletal development in mice. Fibulin-5 is deposited as a fibrous matrix in cranial neural crest-derived mesenchymal tissues, including craniofacial sutures. Fibulin-5-null mice show decreased premaxillary bone outgrowth during postnatal stages. While premaxillo-maxillary suture mesenchymal cells in fibulin-5-null mice were capable of differentiating into osteoblasts, suture cells in mutant mice weremore » less proliferative. Our study provides the first evidence that fibulin-5 is indispensable for the regulation of facial suture mesenchymal cell proliferation required for craniofacial skeletal morphogenesis. - Highlights: • Fibulin-5 is deposited in cranial neural crest-derived mesenchymal tissues. • Fibulin-5-null mice show decreased premaxillary bone growth during postnatal stage. • Fibulin-5 is indispensable for facial suture mesenchymal cell proliferation.« less

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload.

PubMed Central

Nakajima, O; Takahashi, S; Harigae, H; Furuyama, K; Hayashi, N; Sassa, S; Yamamoto, M

1999-01-01

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells. PMID:10562540

Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation.

PubMed

Shimada, Nao; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-09-01

Signal Transducers and Activators of Transcription (STATs) are transcription factors which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells, which are a subset of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones whose gene expression patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes which are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells which is located within the prestalk region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic ecmB gene which is expressed in pstAB cells in parental cells, but which is expressed throughout the prestalk zone in the Dd-STATa-null strain. We also identified several genes which are normally expressed in pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa.

Full-physics 3D heterogeneous simulations of electromagnetic induction fields on level and deformed sea ice

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

Samluk, Jesse P.; Geiger, Cathleen A.; Weiss, Chester J.

In this article we explore simulated responses of electromagnetic (EM) signals relative to in situ field surveys and quantify the effects that different values of conductivity in sea ice have on the EM fields. We compute EM responses of ice types with a three-dimensional (3-D) finite-volume discretization of Maxwell's equations and present 2-D sliced visualizations of their associated EM fields at discrete frequencies. Several interesting observations result: First, since the simulator computes the fields everywhere, each gridcell acts as a receiver within the model volume, and captures the complete, coupled interactions between air, snow, sea ice and sea water asmore » a function of their conductivity; second, visualizations demonstrate how 1-D approximations near deformed ice features are violated. But the most important new finding is that changes in conductivity affect EM field response by modifying the magnitude and spatial patterns (i.e. footprint size and shape) of current density and magnetic fields. These effects are demonstrated through a visual feature we define as 'null lines'. Null line shape is affected by changes in conductivity near material boundaries as well as transmitter location. Our results encourage the use of null lines as a planning tool for better ground-truth field measurements near deformed ice types.« less

Full-physics 3D heterogeneous simulations of electromagnetic induction fields on level and deformed sea ice

DOE PAGES

Samluk, Jesse P.; Geiger, Cathleen A.; Weiss, Chester J.; ...

2015-10-01

In this article we explore simulated responses of electromagnetic (EM) signals relative to in situ field surveys and quantify the effects that different values of conductivity in sea ice have on the EM fields. We compute EM responses of ice types with a three-dimensional (3-D) finite-volume discretization of Maxwell's equations and present 2-D sliced visualizations of their associated EM fields at discrete frequencies. Several interesting observations result: First, since the simulator computes the fields everywhere, each gridcell acts as a receiver within the model volume, and captures the complete, coupled interactions between air, snow, sea ice and sea water asmore » a function of their conductivity; second, visualizations demonstrate how 1-D approximations near deformed ice features are violated. But the most important new finding is that changes in conductivity affect EM field response by modifying the magnitude and spatial patterns (i.e. footprint size and shape) of current density and magnetic fields. These effects are demonstrated through a visual feature we define as 'null lines'. Null line shape is affected by changes in conductivity near material boundaries as well as transmitter location. Our results encourage the use of null lines as a planning tool for better ground-truth field measurements near deformed ice types.« less

Impact of Magnetic Draping, Convection, and Field Line Tying on Magnetopause Reconnection Under Northward IMF

NASA Technical Reports Server (NTRS)

Wendel, Deirdre E.; Reiff, Patricia H.; Goldstein, Melvyn L.

2010-01-01

We simulate a northward IMF cusp reconnection event at the magnetopause using the OpenGGCM resistive MHD code. The ACE input data, solar wind parameters, and dipole tilt belong to a 2002 reconnection event observed by IMAGE and Cluster. Based on a fully three-dimensional skeleton separators, nulls, and parallel electric fields, we show magnetic draping, convection, ionospheric field line tying play a role in producing a series of locally reconnecting nulls with flux ropes. The flux ropes in the cusp along the global separator line of symmetry. In 2D projection, the flux ropes the appearance of a tearing mode with a series of 'x's' and 'o's' but bearing a kind of 'guide field' that exists only within the magnetopause. The reconnecting field lines in the string of ropes involve IMF and both open and closed Earth magnetic field lines. The observed magnetic geometry reproduces the findings of a superposed epoch impact parameter study derived from the Cluster magnetometer data for the same event. The observed geometry has repercussions for spacecraft observations of cusp reconnection and for the imposed boundary conditions reconnection simulations.

Entry of Porphyromonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment.

PubMed

Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-11-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

Entry of Porphyromonas gingivalis Outer Membrane Vesicles into Epithelial Cells Causes Cellular Functional Impairment▿

PubMed Central

Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-01-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis. PMID:19737899

Current singularities at quasi-separatrix layers and three-dimensional magnetic nulls

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

Craig, I. J. D.; Effenberger, Frederic, E-mail: feffen@waikato.ac.nz

2014-11-10

The open problem of how singular current structures form in line-tied, three-dimensional magnetic fields is addressed. A Lagrangian magneto-frictional relaxation method is employed to model the field evolution toward the final near-singular state. Our starting point is an exact force-free solution of the governing magnetohydrodynamic equations that is sufficiently general to allow for topological features like magnetic nulls to be inside or outside the computational domain, depending on a simple set of parameters. Quasi-separatrix layers (QSLs) are present in these structures and, together with the magnetic nulls, they significantly influence the accumulation of current. It is shown that perturbations affectingmore » the lateral boundaries of the configuration lead not only to collapse around the magnetic null but also to significant QSL currents. Our results show that once a magnetic null is present, the developing currents are always attracted to that specific location and show a much stronger scaling with resolution than the currents that form along the QSL. In particular, the null-point scalings can be consistent with models of 'fast' reconnection. The QSL currents also appear to be unbounded but give rise to weaker singularities, independent of the perturbation amplitude.« less

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress.

PubMed

von Einem, Jens; Schumacher, Daniel; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2006-03-01

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status.

PubMed

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G; Wong, Kwok-Kin; Khuri, Fadlo R; Zhou, Wei; Shin, Dong M

2017-08-29

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 ( kras G12D/wt /p53 -/- /lkb1 wt/wt ) and t2 ( kras G12D/wt /p53 -/- / lkb1 -/- ) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer.

Expression and function of orphan nuclear receptor TLX in adult neural stem cells.

PubMed

Shi, Yanhong; Chichung Lie, D; Taupin, Philippe; Nakashima, Kinichi; Ray, Jasodhara; Yu, Ruth T; Gage, Fred H; Evans, Ronald M

2004-01-01

The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. TLX was initially identified as an orphan nuclear receptor expressed in vertebrate forebrains and is highly expressed in the adult brain. The brains of TLX-null mice have been reported to have no obvious defects during embryogenesis; however, mature mice suffer from retinopathies, severe limbic defects, aggressiveness, reduced copulation and progressively violent behaviour. Here we show that TLX maintains adult neural stem cells in an undifferentiated, proliferative state. We show that TLX-expressing cells isolated by fluorescence-activated cell sorting (FACS) from adult brains can proliferate, self-renew and differentiate into all neural cell types in vitro. By contrast, TLX-null cells isolated from adult mutant brains fail to proliferate. Reintroducing TLX into FACS-sorted TLX-null cells rescues their ability to proliferate and to self-renew. In vivo, TLX mutant mice show a loss of cell proliferation and reduced labelling of nestin in neurogenic areas in the adult brain. TLX can silence glia-specific expression of the astrocyte marker GFAP in neural stem cells, suggesting that transcriptional repression may be crucial in maintaining the undifferentiated state of these cells.

Characterization of the spontaneous DNA-synthesizing and/or IgG-secreting cells in peripheral blood from patients with systemic lupus erythematosus

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

Yano, K.; Morimasa, K.; Asano, T.

1985-04-01

Characterization of the cells responsible for spontaneous DNA synthesis and/or IgG secretion in systemic lupus erythematosus (SLE) was undertaken by fractionation of the peripheral blood mononuclear cells (PBM). Each fraction was analyzed for its capacity to incorporate (/sup 3/H)thymidine ((/sup 3/H)TdR) and secrete IgG without mitogen. The non-E rosette-forming cell (non-ERRC) fraction, which consisted of the surface immunoglobulin-positive (sIg(+)) cells and null cells, revealed a markedly increased spontaneous DNA synthesis (620.0 +/- 586.9 cpm) during the first hour of culture and an elevated spontaneous IgG secretion (8639 +/- 2630 ng/ml) during 9 days of culture. Of particular interest was themore » finding that both increased responses were conducted by the null cells; the null cell population had approximately a fourfold relative increase of (/sup 3/H)TdR incorporation and a 60-fold relative increase of IgG secretion compared with the sIg(+) cell population. These results suggest that SLE patients have a small population of preactivated B-cell lineage cells, which lack sIg or have a very low density of sIg.« less

Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV-deficient B cells.

PubMed

Han, Li; Yu, Kefei

2008-11-24

Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4-null cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.

Inhibition of PTEN and activation of Akt by menadione.

PubMed

Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

2007-04-01

Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

A critical role for the transcription factor Scl in platelet production during stress thrombopoiesis.

PubMed

McCormack, Matthew P; Hall, Mark A; Schoenwaelder, Simone M; Zhao, Quan; Ellis, Sarah; Prentice, Julia A; Clarke, Ashleigh J; Slater, Nicholas J; Salmon, Jessica M; Jackson, Shaun P; Jane, Stephen M; Curtis, David J

2006-10-01

The generation of platelets from megakaryocytes in the steady state is regulated by a variety of cytokines and transcription factors, including thrombopoietin (TPO), GATA-1, and NF-E2. Less is known about platelet production in the setting of stress thrombopoiesis, a pivotal event in the context of cytotoxic chemotherapy. Here we show in mice that the transcription factor Scl is critical for platelet production after chemotherapy and in thrombopoiesis induced by administration of TPO. Megakaryocytes from these mice showed appropriate increases in number and ploidy but failed to shed platelets. Ultrastructural examination of Scl-null megakaryocytes revealed a disorganized demarcation membrane and reduction in platelet granules. Quantitative real-time polymerase chain reaction showed that Scl-null platelets lacked NF-E2, and chromatin immunoprecipitation analysis demonstrated Scl binding to the NF-E2 promoter in the human megakaryoblastic-cell line Meg-01, along with its binding partners E47, Lmo2, and the cofactors Ldb1 and GATA-2. These findings suggest that Scl acts up-stream of NF-E2 expression to control megakaryocyte development and platelet release in settings of thrombopoietic stress.

Laron Dwarfism and Non-Insulin-Dependent Diabetes Mellitus in the Hnf-1α Knockout Mouse

PubMed Central

Lee, Ying-Hue; Sauer, Brian; Gonzalez, Frank J.

1998-01-01

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1α) were produced by use of the Cre-loxP recombination system. HNF-1α-null mice are viable but sterile and exhibit a phenotype reminiscent of both Laron-type dwarfism and non-insulin-dependent diabetes mellitus (NIDDM). In contrast to an earlier HNF-1α-null mouse line that had been produced by use of standard gene disruption methodology (M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. P. Bach, C. Babinet, and M. Yaniv, Cell 84:575–585, 1996), these mice exhibited no increased mortality and only minimal renal dysfunction during the first 6 months of development. Both dwarfism and NIDDM are most likely due to the loss of expression of insulin-like growth factor I (IGF-I) and lower levels of insulin, resulting in stunted growth and elevated serum glucose levels, respectively. These results confirm the functional significance of the HNF-1α regulatory elements that had previously been shown to reside in the promoter regions of both the IGF-I and the insulin genes. PMID:9566924

Role of RANKL (TNFSF11)-dependent osteopetrosis in the dental phenotype of Msx2 null mutant mice.

PubMed

Castaneda, Beatriz; Simon, Yohann; Ferbus, Didier; Robert, Benoit; Chesneau, Julie; Mueller, Christopher; Berdal, Ariane; Lézot, Frédéric

2013-01-01

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 (-/-)) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 (-/-) mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 (-/-) mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 (-/-) Rank(Tg) mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 (-/-) mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor.

Magnetoacoustic Waves in a Stratified Atmosphere with a Magnetic Null Point

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

Tarr, Lucas A.; Linton, Mark; Leake, James, E-mail: lucas.tarr.ctr@nrl.navy.mil

2017-03-01

We perform nonlinear MHD simulations to study the propagation of magnetoacoustic waves from the photosphere to the low corona. We focus on a 2D system with a gravitationally stratified atmosphere and three photospheric concentrations of magnetic flux that produce a magnetic null point with a magnetic dome topology. We find that a single wavepacket introduced at the lower boundary splits into multiple secondary wavepackets. A portion of the packet refracts toward the null owing to the varying Alfvén speed. Waves incident on the equipartition contour surrounding the null, where the sound and Alfvén speeds coincide, partially transmit, reflect, and mode-convertmore » between branches of the local dispersion relation. Approximately 15.5% of the wavepacket’s initial energy ( E {sub input}) converges on the null, mostly as a fast magnetoacoustic wave. Conversion is very efficient: 70% of the energy incident on the null is converted to slow modes propagating away from the null, 7% leaves as a fast wave, and the remaining 23% (0.036 E {sub input}) is locally dissipated. The acoustic energy leaving the null is strongly concentrated along field lines near each of the null’s four separatrices. The portion of the wavepacket that refracts toward the null, and the amount of current accumulation, depends on the vertical and horizontal wavenumbers and the centroid position of the wavepacket as it crosses the photosphere. Regions that refract toward or away from the null do not simply coincide with regions of open versus closed magnetic field or regions of particular field orientation. We also model wavepacket propagation using a WKB method and find that it agrees qualitatively, though not quantitatively, with the results of the numerical simulation.« less

High frequency generation in the corona: Resonant cavities

NASA Astrophysics Data System (ADS)

Santamaria, I. C.; Van Doorsselaere, T.

2018-03-01

Aims: Null points are prominent magnetic field singularities in which the magnetic field strength strongly decreases in very small spatial scales. Around null points, predicted to be ubiquitous in the solar chromosphere and corona, the wave behavior changes considerably. Null points are also responsible for driving very energetic phenomena, and for contributing to chromospheric and coronal heating. In previous works we demonstrated that slow magneto-acoustic shock waves were generated in the chromosphere propagate through the null point, thereby producing a train of secondary shocks escaping along the field lines. A particular combination of the shock wave speeds generates waves at a frequency of 80 MHz. The present work aims to investigate this high frequency region around a coronal null point to give a plausible explanation to its generation at that particular frequency. Methods: We carried out a set of two-dimensional numerical simulations of wave propagation in the neighborhood of a null point located in the corona. We varied both the amplitude of the driver and the atmospheric properties to investigate the sensitivity of the high frequency waves to these parameters. Results: We demonstrate that the wave frequency is sensitive to the atmospheric parameters in the corona, but it is independent of the strength of the driver. Thus, the null point behaves as a resonant cavity generating waves at specific frequencies that depend on the background equilibrium model. Moreover, we conclude that the high frequency wave train generated at the null point is not necessarily a result of the interaction between the null point and a shock wave. This wave train can be also developed by the interaction between the null point and fast acoustic-like magneto-acoustic waves, that is, this interaction within the linear regime.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

PubMed Central

Foltz, Jennifer A.; Somanchi, Srinivas S.; Yang, Yanwen; Aquino-Lopez, Arianexys; Bishop, Erin E.; Lee, Dean A.

2016-01-01

Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy. PMID:27933061

Null hypersurfaces in de Sitter and anti-de Sitter cosmologies

NASA Astrophysics Data System (ADS)

Hogan, P. A.

The study of gravitational waves in the presence of a cosmological constant has led to interesting forms of the de Sitter and anti-de Sitter line elements based on families of null hypersurfaces. The forms are interesting because they focus attention on the geometry of null hypersurfaces in spacetimes of constant curvature. Two examples are worked out in some detail. The first originated in the study of collisions of impulsive gravitational waves in which the post-collision spacetime is a solution of Einstein’s field equations with a cosmological constant, and the second originated in the generalization of plane fronted gravitational waves with parallel rays to include a cosmological constant.

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

PubMed Central

Ruas, Margarida; Davis, Lianne C; Chen, Cheng-Chang; Morgan, Anthony J; Chuang, Kai-Ting; Walseth, Timothy F; Grimm, Christian; Garnham, Clive; Powell, Trevor; Platt, Nick; Platt, Frances M; Biel, Martin; Wahl-Schott, Christian; Parrington, John; Galione, Antony

2015-01-01

The second messenger NAADP triggers Ca2+ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca2+ responses as assessed by single-cell Ca2+ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca2+-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca2+ release. High-affinity [32P]NAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+-permeable channels indispensable for NAADP signalling. PMID:25872774

Histone h1 depletion impairs embryonic stem cell differentiation.

PubMed

Zhang, Yunzhe; Cooke, Marissa; Panjwani, Shiraj; Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T; Pan, Chenyi; McDevitt, Todd C; Fan, Yuhong

2012-01-01

Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes.

High-throughput drug screen identifies chelerythrine as a selective inducer of death in a TSC2-null setting.

PubMed

Medvetz, Doug; Sun, Yang; Li, Chenggang; Khabibullin, Damir; Balan, Murugabaskar; Parkhitko, Andrey; Priolo, Carmen; Asara, John M; Pal, Soumitro; Yu, Jane; Henske, Elizabeth P

2015-01-01

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome associated with tumors of the brain, heart, kidney, and lung. The TSC protein complex inhibits the mammalian or mechanistic target of rapamycin complex 1 (mTORC1). Inhibitors of mTORC1, including rapamycin, induce a cytostatic response in TSC tumors, resulting in temporary disease stabilization and prompt regrowth when treatment is stopped. The lack of TSC-specific cytotoxic therapies represents an important unmet clinical need. Using a high-throughput chemical screen in TSC2-deficient, patient-derived cells, we identified a series of molecules antagonized by rapamycin and therefore selective for cells with mTORC1 hyperactivity. In particular, the cell-permeable alkaloid chelerythrine induced reactive oxygen species (ROS) and depleted glutathione (GSH) selectively in TSC2-null cells based on metabolic profiling. N-acetylcysteine or GSH cotreatment protected TSC2-null cells from chelerythrine's effects, indicating that chelerythrine-induced cell death is ROS dependent. Induction of heme-oxygenase-1 (HMOX1/HO-1) with hemin also blocked chelerythrine-induced cell death. In vivo, chelerythrine inhibited the growth of TSC2-null xenograft tumors with no evidence of systemic toxicity with daily treatment over an extended period of time. This study reports the results of a bioactive compound screen and the identification of a potential lead candidate that acts via a novel oxidative stress-dependent mechanism to selectively induce necroptosis in TSC2-deficient tumors. This study demonstrates that TSC2-deficient tumor cells are hypersensitive to oxidative stress-dependent cell death, and provide critical proof of concept that TSC2-deficient cells can be therapeutically targeted without the use of a rapalog to induce a cell death response. ©2014 American Association for Cancer Research.

MMP20 Promotes a Smooth Enamel Surface, a Strong DEJ, and a Decussating Enamel Rod Pattern

PubMed Central

Bartlett, John D.; Skobe, Ziedonis; Nanci, Antonio; Smith, Charles E.

2012-01-01

Mutations of the Matrix metalloproteinase-20 (MMP20, enamelysin) gene cause autosomal recessive amelogenesis imperfecta and Mmp20 ablated mice also have malformed dental enamel. Here we show that Mmp20 null mouse secretory stage ameloblasts maintained a columnar shape and were present as a single layer of cells. However, the null maturation stage ameloblasts covered extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin indicative of a faulty DEJ. The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The Mmp20 null mouse enamel rods were grossly malformed or were absent indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ. PMID:22243247

pyNSMC: A Python Module for Null-Space Monte Carlo Uncertainty Analysis

NASA Astrophysics Data System (ADS)

White, J.; Brakefield, L. K.

2015-12-01

The null-space monte carlo technique is a non-linear uncertainty analyses technique that is well-suited to high-dimensional inverse problems. While the technique is powerful, the existing workflow for completing null-space monte carlo is cumbersome, requiring the use of multiple commandline utilities, several sets of intermediate files and even a text editor. pyNSMC is an open-source python module that automates the workflow of null-space monte carlo uncertainty analyses. The module is fully compatible with the PEST and PEST++ software suites and leverages existing functionality of pyEMU, a python framework for linear-based uncertainty analyses. pyNSMC greatly simplifies the existing workflow for null-space monte carlo by taking advantage of object oriented design facilities in python. The core of pyNSMC is the ensemble class, which draws and stores realized random vectors and also provides functionality for exporting and visualizing results. By relieving users of the tedium associated with file handling and command line utility execution, pyNSMC instead focuses the user on the important steps and assumptions of null-space monte carlo analysis. Furthermore, pyNSMC facilitates learning through flow charts and results visualization, which are available at many points in the algorithm. The ease-of-use of the pyNSMC workflow is compared to the existing workflow for null-space monte carlo for a synthetic groundwater model with hundreds of estimable parameters.

Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules.

PubMed

Clark, A M; Garland, K K; Russell, L D

2000-12-01

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

Lymphocyte senescence in COPD is associated with decreased histone deacetylase 2 expression by pro-inflammatory lymphocytes.

PubMed

Hodge, Greg; Jersmann, Hubertus; Tran, Hai B; Roscioli, Eugene; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2015-10-24

Histone acetyltransferases (HAT) and histone deacetylases (HDAC) are enzymes that upregulate and down-regulate pro-inflammatory gene transcription respectively. HDAC2 is required by corticosteroids to switch off activated inflammatory genes and is reduced in lung macrophages in COPD. We have shown that COPD patients have increased steroid resistant CD28null (senescent) pro-inflammatory T and NKT-like peripheral blood cells (particularly CD8+ subsets) and we hypothesized that these changes would be associated with a loss of HDAC2 from these senescent pro-inflammatory lymphocytes. Blood was collected from 10 COPD and 10 aged-matched controls. Intracellular pro-inflammatory cytokines, IFNγ and TNFα, and expression of CD28, HDAC2 and HAT, were determined in lymphocyte subsets in the presence of ± 5 mg/ml theophylline (HDAC2 activator), 10 μM prednisolone and 2.5 ng/ml cyclosporine A (immunosuppressant), using flow cytometry. There was a loss of HDAC2 from CD28null CD8+ T and NKT-like cells in COPD. There was a significant negative correlation between HDAC2 expression and the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -.763, p < 0.001 for T-cell IFNγ). There was a synergistic upregulation of HDAC2 and associated decrease in pro-inflammatory cytokine production in CD28nullCD8+ T and NKT-like cells in the presence of 5 mg/L theophylline + 10(-6) M prednisolone or 2.5 ng/mL cyclosporine A (CsA). Lymphocyte senescence in COPD is associated with loss of HDAC2 in CD28nullCD8+ T and NKT-like cells. Alternative treatment options such as combined theophylline with low-dose CsA, that inhibit these pro-inflammatory cells, may reduce systemic inflammation in COPD.

Steroid resistance in COPD is associated with impaired molecular chaperone Hsp90 expression by pro-inflammatory lymphocytes.

PubMed

Hodge, Greg; Roscioli, Eugene; Jersmann, Hubertus; Tran, Hai B; Holmes, Mark; Reynolds, Paul N; Hodge, Sandra

2016-10-21

Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 μM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 μM prednisolone and 2.5 ng/mL cyclosporine A. Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.

Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

PubMed Central

Hu, Shuiying; Niu, Hongmei; Inaba, Hiroto; Orwick, Shelley; Rose, Charles; Panetta, John C.; Yang, Shengping; Pounds, Stanley; Fan, Yiping; Calabrese, Christopher; Rehg, Jerold E.; Campana, Dario; Rubnitz, Jeffrey E.

2011-01-01

Background Acute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy. Methods Using 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγnull mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration–time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγnull mice bearing AML xenografts. Results Sorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P < .001 for combination twice daily vs all other treatments listed). Conclusions Sorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML. PMID:21487100

VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

PubMed Central

Morini, Marco F.; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I.; Conze, Lei L.; O’Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P.

2018-01-01

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. PMID:29233846

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

PubMed

Morini, Marco F; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I; Conze, Lei L; O'Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P; Dejana, Elisabetta; Taddei, Andrea

2018-01-19

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. © 2017 The Authors.

Induction and persistence of abnormal testicular germ cells following gestational exposure to di-(n-butyl) phthalate in p53-null mice.

PubMed

Saffarini, Camelia M; Heger, Nicholas E; Yamasaki, Hideki; Liu, Tao; Hall, Susan J; Boekelheide, Kim

2012-01-01

Phthalate esters are commonly used plasticizers found in many household items, personal care products, and medical devices. Animal studies have shown that in utero exposure to di-(n-butyl) phthalate (DBP) within a critical window during gestation causes male reproductive tract abnormalities resembling testicular dysgenesis syndrome. Our studies utilized p53-deficient mice for their ability to display greater resistance to apoptosis during development. This model was chosen to determine whether multinucleated germ cells (MNG) induced by gestational DBP exposure could survive postnatally and evolve into testicular germ cell cancer. Pregnant dams were exposed to DBP (500 mg/kg/day) by oral gavage from gestational day 12 until birth. Perinatal effects were assessed on gestational day 19 and postnatal days 1, 4, 7, and 10 for the number of MNGs present in control and DBP-treated p53-heterozygous and null animals. As expected, DBP exposure induced MNGs, with greater numbers found in p53-null mice. Additionally, there was a time-dependent decrease in the incidence of MNGs during the early postnatal period. Histologic examination of adult mice exposed in utero to DBP revealed persistence of abnormal germ cells only in DBP-treated p53-null mice, not in p53-heterozygous or wild-type mice. Immunohistochemical staining of perinatal MNGs and adult abnormal germ cells was negative for both octamer-binding protein 3/4 and placental alkaline phosphatase. This unique model identified a role for p53 in the perinatal apoptosis of DBP-induced MNGs and provided insight into the long-term effects of gestational DBP exposure within a p53-null environment.

Emv30null NOD-scid mice. An improved host for adoptive transfer of autoimmune diabetes and growth of human lymphohematopoietic cells.

PubMed

Serreze, D V; Leiter, E H; Hanson, M S; Christianson, S W; Shultz, L D; Hesselton, R M; Greiner, D L

1995-12-01

When used as hosts in passive transfer experiments, a stock of NOD/Lt mice congenic for the severe combined immunodeficiency (scid) mutation have provided great insight to the contributions of various T-cell populations in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). Moreover, NOD-scid mice support higher levels of human lymphohematopoietic cell growth than the C.B-17-scid strain in which the mutation originated. However, the ability to perform long-term lymphohematopoietic repopulation studies in the NOD-scid stock has been limited by the fact that most of these mice develop lethal thymic lymphomas beginning at 20 weeks of age. These thymic lymphomas are characterized by activation and subsequent genomic reintegrations of Emv30, an endogenous murine ecotropic retrovirus unique to the NOD genome. To test the role of this endogenous retrovirus in thymomagenesis, we produced a stock of Emv30null NOD-scid mice by congenic replacement of the proximal end of chromosome 11 with genetic material derived from the closely related NOR/Lt strain. Thymic lymphomas still initiate in Emv30null NOD-scid females, but their rate of progression is significantly retarded since the frequency of tumors weighing between 170 and 910 mg at 25 weeks of age was reduced to 20.8% vs. 76.2% in Emv30% segregants. The thymic lymphomas that did develop in Emv30null NOD-scid mice were not characterized by a compensatory increase in mink cell focus-forming proviral integrations, which initiate thymomagenesis in other susceptible mouse strains. Significantly, the ability of standard NOD T-cells to transfer IDDM to the Emv30null NOD-scid stock was not impaired.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881

TM4SF5 promotes metastatic behavior of cells in 3D extracellular matrix gels by reducing dependency on environmental cues

PubMed Central

Nam, Seo Hee; Cheong, Jin-Gyu; Jeong, Doyoung; Lee, Seo-Jin; Pan, Cheol-Ho; Jung, Jae Woo; Kim, Hye-Jin; Ryu, Jihye; Kim, Ji Eon; Kim, Somi; Cho, Chang Yun; Kang, Min-Kyung; Lee, Kyung-Min; Lee, Jung Weon

2017-01-01

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocellular carcinoma tissues and enhances migration in two-dimensional environments. Here, we investigated how TM4SF5 is involved in diverse pro-metastatic phenotypes in in vivo-like three-dimensional (3D) extracellular matrix gels. TM4SF5-positive cells aggressively formed invasive foci in 3D Matrigel, depending on TM4SF5-mediated signaling activity, cytoskeletal organization, and matrix metallopeptidase (MMP) 2-mediated extracellular remodeling, whereas TM4SF5-null cells did not. The TM4SF5-null cells did, however, form invasive foci in 3D Matrigel following inhibition of Rho-associated protein kinase or addition of collagen I, suggesting that collagen I compensated for TM4SF5 expression. Similarly, TM4SF5-positive cells expressing vascular endothelial-cadherin formed network-like vasculogenic mimicry in 3D Matrigel and collagen I mixture gels, whereas TM4SF5-negative cells in the mixture gels displayed the network structures only upon further treatment with epidermal growth factor. The foci formation also required MMP2-mediated remodeling of the extracellular matrix. Co-cultures exhibited TM4SF5-positive or cancer-associated fibroblasts at the outward edges of TM4SF5-null cell clusters. Compared with TM4SF5-null cells, TM4SF5-positive cells in 3D collagen gels showed a more invasive outgrowth with dramatic invadopodia. These observations suggest that TM4SF5 plays roles in the promotion of diverse metastatic properties with fewer environmental requirements than TM4SF5-negative cells. PMID:29137358

Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

PubMed

del Río, A; Barrio, M C; Murillo, J; Maldonado, E; López-Gordillo, Y; Martínez-Sanz, E; Martínez, M L; Martínez-Álvarez, C

2011-01-01

The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed. Copyright © 2010 S. Karger AG, Basel.

Reduced wheel running and blunted effects of voluntary exercise in LPA1-null mice: The importance of assessing the amount of running in transgenic mice studies

PubMed Central

Castilla-Ortega, Estela; Rosell-Valle, Cristina; Blanco, Eduardo; Pedraza, Carmen; Chun, Jerold; de Fonseca, Fernando Rodríguez; Estivill-Torrús, Guillermo; Santín, Luis J.

2014-01-01

This work was aimed to assess whether voluntary exercise rescued behavioral and hippocampal alterations in mice lacking the lysophosphatidic acid LPA1 receptor (LPA1-null mice), studying the potential relationship between the amount of exercise performed and its effects. Normal and LPA1-null mice underwent 23 days of free wheel running and were tested for open-field behavior and adult hippocampal neurogenesis (cell proliferation, immature neurons, cell survival). Running decreased anxiety-like behavior in both genotypes but increased exploration only in the normal mice. While running affected all neurogenesis-related measures in normal mice (especially in the suprapyramidal blade of the dentate gyrus), only a moderate increase in cell survival was found in the mutants. Importantly, the LPA1-nulls showed notably reduced running. Analysis suggested that defective running in the LPA1-null mice could contribute to explain the scarce benefit of the voluntary exercise treatment. On the other hand, a literature review revealed that voluntary exercise is frequently used to modulate behavior and the hippocampus in transgenic mice, but half of the studies did not assess the quantity of running, overlooking any potential running impairments. This study adds evidence to the relevance of the quantity of exercise performed, emphasizing the importance of its assessment in transgenic mice research. PMID:24055600

Severe changes in colon epithelium in the Mecp2-null mouse model of Rett syndrome.

PubMed

Millar-Büchner, Pamela; Philp, Amber R; Gutierrez, Noemí; Villanueva, Sandra; Kerr, Bredford; Flores, Carlos A

2016-12-01

Rett syndrome is best known due to its severe and devastating symptoms in the central nervous system. It is produced by mutations affecting the Mecp2 gene that codes for a transcription factor. Nevertheless, evidence for MECP2 activity has been reported for tissues other than those of the central nervous system. Patients affected by Rett presented with intestinal affections whose origin is still not known. We have observed that the Mecp2-null mice presented with episodes of diarrhea, and decided to study the intestinal phenotype in these mice. Mecp2-null mice or bearing the conditional intestinal deletion of MECP2 were used. Morphometirc and histologic analysis of intestine, and RT-PCR, western blot and immunodetection were perfomed on intestinal samples of the animals. Electrical parameters of the intestine were determined by Ussing chamber experiments in freshly isolated colon samples. First we determined that MECP2 protein is mainly expressed in cells of the lower part of the colonic crypts and not in the small intestine. The colon of the Mecp2-null mice was shorter than that of the wild-type. Histological analysis showed that epithelial cells of the surface have abnormal localization of key membrane proteins like ClC-2 and NHE-3 that participate in the electroneutral NaCl absorption; nevertheless, electrogenic secretion and absorption remain unaltered. We also detected an increase in a proliferation marker in the crypts of the colon samples of the Mecp2-null mice, but the specific silencing of Mecp2 from intestinal epithelium was not able to recapitulate the intestinal phenotype of the Mecp2-null mice. In summary, we showed that the colon is severely affected by Mecp2 silencing in mice. Changes in colon length and epithelial histology are similar to those observed in colitis. Changes in the localization of proteins that participate in fluid absorption can explain watery stools, but the exclusive deletion of Mecp2 from the intestine did not reproduce colon changes observed in the Mecp2-null mice, indicating the participation of other cells in this phenotype and the complex interaction between different cell types in this disease.

Epidermal growth factor impairs palatal shelf adhesion and fusion in the Tgf-β 3 null mutant.

PubMed

Barrio, M Carmen; Del Río, Aurora; Murillo, Jorge; Maldonado, Estela; López-Gordillo, Yamila; Paradas-Lara, Irene; Hernandes, Luzmarina; Catón, Javier; Martínez-Álvarez, Concepción

2014-01-01

The cleft palate presented by transforming growth factor-β3 (Tgf-β3) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 (Tgf-β1) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β1 and Msx-1 in Tgf-β3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal mesenchyme. Inhibition of TGF-β1 does not affect either EGF or Msx-1 expression. © 2014 S. Karger AG, Basel.

Loss of α-calcitonin gene-related peptide (αCGRP) reduces the efficacy of the Vestibulo-ocular Reflex (VOR).

PubMed

Luebke, Anne E; Holt, Joseph C; Jordan, Paivi M; Wong, Yi Shan; Caldwell, Jillian S; Cullen, Kathleen E

2014-07-30

The neuroactive peptide calcitonin-gene related peptide (CGRP) is known to act at efferent synapses and their targets in hair cell organs, including the cochlea and lateral line. CGRP is also expressed in vestibular efferent neurons as well as a number of central vestibular neurons. Although CGRP-null (-/-) mice demonstrate a significant reduction in cochlear nerve sound-evoked activity compared with wild-type mice, it is unknown whether and how the loss of CGRP influence vestibular system function. Vestibular function was assessed by quantifying the vestibulo-ocular reflex (VOR) in alert mice. The loss of CGRP in (-/-) mice was associated with a reduction of the VOR gain of ≈50% without a concomitant change in phase. Using immunohistochemistry, we confirmed that, although CGRP staining was absent in the vestibular end-organs of null (-/-) mice, cholinergic staining appeared normal, suggesting that the overall gross development of vestibular efferent innervation was unaltered. We further confirmed that the observed deficit in vestibular function of null (-/-) mice was not the result of nontargeted effects at the level of the extraocular motor neurons and/or their innervation of extraocular muscles. Analysis of the relationship between vestibular quick phase amplitude and peak velocity revealed that extraocular motor function was unchanged, and immunohistochemistry revealed no abnormalities in motor endplates. Together, our findings show that the neurotransmitter CGRP plays a key role in ensuring VOR efficacy. Copyright © 2014 the authors 0270-6474/14/3410453-06$15.00/0.

IGF-1-overexpressing mesenchymal stem cells accelerate bone marrow stem cell mobilization via paracrine activation of SDF-1alpha/CXCR4 signaling to promote myocardial repair.

PubMed

Haider, Husnain Kh; Jiang, Shujia; Idris, Niagara M; Ashraf, Muhammad

2008-11-21

We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like growth factor (IGF)-1 showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through paracrine release of stromal cell-derived factor (SDF)-1alpha. Rat bone marrow-derived MSCs were used as nontransduced ((Norm)MSCs) or transduced with adenoviral-null vector ((Null)MSCs) or vector encoding for IGF-1 ((IGF-1)MSCs). (IGF-1)MSCs secreted higher IGF-1 until 12 days of observation (P<0.001 versus (Null)MSCs). Molecular studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl.xL and inhibition of glycogen synthase kinase 3beta besides release of SDF-1alpha in parallel with IGF-1 expression in (IGF-1)MSCs. For in vivo studies, 70 muL of DMEM without cells (group 1) or containing 1.5x10(6) (Null)MSCs (group 2) or (IGF-1)MSCs (group 3) were implanted intramyocardially in a female rat model of permanent coronary artery occlusion. One week later, immunoblot on rat heart tissue (n=4 per group) showed elevated myocardial IGF-1 and phospho-Akt in group 3 and higher survival of (IGF-1)MSCs (P<0.06 versus (Null)MSCs) (n=6 per group). SDF-1alpha was increased in group 3 animal hearts (20-fold versus group 2), with massive mobilization and homing of ckit(+), MDR1(+), CD31(+), and CD34(+) cells into the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Confocal imaging after immunostaining for myosin heavy chain, actinin, connexin-43, and von Willebrand factor VIII showed extensive angiomyogenesis in the infarcted heart. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 3 as compared with group 1 (P<0.05). In conclusion, the strategy of IGF-1 transgene expression induced massive stem cell mobilization via SDF-1alpha signaling and culminated in extensive angiomyogenesis in the infarcted heart.

Balloon Exoplanet Nulling Interferometer (BENI)

NASA Technical Reports Server (NTRS)

Lyon, Richard G.; Clampin, Mark; Woodruff, Robert A.; Vasudevan, Gopal; Ford, Holland; Petro, Larry; Herman, Jay; Rinehart, Stephen; Carpenter, Kenneth; Marzouk, Joe

2009-01-01

We evaluate the feasibility of using a balloon-borne nulling interferometer to detect and characterize exosolar planets and debris disks. The existing instrument consists of a 3-telescope Fizeau imaging interferometer with 3 fast steering mirrors and 3 delay lines operating at 800 Hz for closed-loop control of wavefront errors and fine pointing. A compact visible nulling interferometer is under development which when coupled to the imaging interferometer would in-principle allow deep suppression of starlight. We have conducted atmospheric simulations of the environment above 100,000 feet and believe balloons are a feasible path forward towards detection and characterization of a limited set of exoplanets and their debris disks. Herein we will discuss the BENI instrument, the balloon environment and the feasibility of such as mission.

Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase

PubMed Central

Ng, Ho Yin; Oliver, Brian Gregory George; Burgess, Janette Kay; Krymskaya, Vera P; Black, Judith Lee; Moir, Lyn M

2015-01-01

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2–59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction. PMID:26282580

Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-β1/FAK/ERK pathway.

PubMed

Jahan, Rahat; Macha, Muzafar A; Rachagani, Satyanarayana; Das, Srustidhar; Smith, Lynette M; Kaur, Sukhwinder; Batra, Surinder K

2018-08-01

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC. Copyright © 2018 Elsevier B.V. All rights reserved.

Formation of the Sun-aligned arc region and the void (polar slot) under the null-separator structure

NASA Astrophysics Data System (ADS)

Tanaka, T.; Obara, T.; Watanabe, M.; Fujita, S.; Ebihara, Y.; Kataoka, R.

2017-04-01

From the global magnetosphere-ionosphere coupling simulation, we examined the formation of the Sun-aligned arc region and the void (polar slot) under the northward interplanetary magnetic field (IMF) with negative By condition. In the magnetospheric null-separator structure, the separatrices generated from two null points and two separators divide the entire space into four types of magnetic region, i.e., the IMF, the northern open magnetic field, the southern open magnetic field, and the closed magnetic field. In the ionosphere, the Sun-aligned arc region and the void are reproduced in the distributions of simulated plasma pressure and field-aligned current. The outermost closed magnetic field lines on the boundary (separatrix) between the northern open magnetic field and the closed magnetic field are projected to the northern ionosphere at the boundary between the Sun-aligned arc region and the void, both on the morning and evening sides. The magnetic field lines at the plasma sheet inner edge are projected to the equatorward boundary of the oval. Therefore, the Sun-aligned arc region is on the closed magnetic field lines of the plasma sheet. In the plasma sheet, an inflated structure (bulge) is generated at the junction of the tilted plasma sheet in the far-to-middle tail and nontilted plasma sheet in the ring current region. In the Northern Hemisphere, the bulge is on the evening side wrapped by the outermost closed magnetic field lines that are connected to the northern evening ionosphere. This inflated structure (bulge) is associated with shear flows that cause the Sun-aligned arc.

Parameterization of spectral baseline directly from short echo time full spectra in 1 H-MRS.

PubMed

Lee, Hyeong Hun; Kim, Hyeonjin

2017-09-01

To investigate the feasibility of parameterizing macromolecule (MM) resonances directly from short echo time (TE) spectra rather than pre-acquired, T 1 -weighted, metabolite-nulled spectra in 1 H-MRS. Initial line parameters for metabolites and MMs were set for rat brain spectra acquired at 9.4 Tesla upon a priori knowledge. Then, MM line parameters were optimized over several steps with fixed metabolite line parameters. The proposed method was tested by estimating metabolite T 1 . The results were compared with those obtained with two existing methods. Furthermore, subject-specific, spin density-weighted, MM model spectra were generated according to the MM line parameters from the proposed method for metabolite quantification. The results were compared with those obtained with subject-specific, T 1 -weighted, metabolite-nulled spectra. The metabolite T 1 were largely in close agreement among the three methods. The spin density-weighted MM resonances from the proposed method were in good agreement with the T 1 -weighted, metabolite-nulled spectra except for the MM resonance at ∼3.2 ppm. The metabolite concentrations estimated by incorporating these two different spectral baselines were also in good agreement except for several metabolites with resonances at ∼3.2 ppm. The MM parameterization directly from short-TE spectra is feasible. Further development of the method may allow for better representation of spectral baseline with negligible T 1 -weighting. Magn Reson Med 78:836-847, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Six post-implantation lethal knockouts of genes for lipophilic MAPK pathway proteins are expressed in preimplantation mouse embryos and trophoblast stem cells.

PubMed

Xie, Yufen; Wang, Yingchun; Sun, Tong; Wang, Fangfei; Trostinskaia, Anna; Puscheck, Elizabeth; Rappolee, Daniel A

2005-05-01

Mitogen-activated protein kinase (MAPK) signaling pathways play an important role in controlling embryonic proliferation and differentiation. It has been demonstrated that sequential lipophilic signal transduction mediators that participate in the MAPK pathway are null post-implantation lethal. It is not clear why the lethality of these null mutants arises after implantation and not before. One hypothesis is that the gene product of these post-implantation lethal null mutants are not present before implantation in normal embryos and do not have function until after implantation. To test this hypothesis, we selected a set of lipophilic genes mediating MAPK signal transduction pathways whose null mutants result in early peri-implantation or placental lethality. These included FRS2alpha, GAB1, GRB2, SOS1, Raf-B, and Raf1. Products of these selected genes were detected and their locations and functions indicated by indirect immunocytochemistry and Western blotting for proteins and RT-polymerase chain reaction (PCR) for mRNA transcription. We report here that all six signal mediators are detected at the protein level in preimplantation mouse embryo, placental trophoblasts, and in cultured trophoblast stem cells (TSC). Proteins are all detected in E3.5 embryos at a time when the first known mitogenic intercellular communication has been documented. mRNA transcripts of two post-implantation null mutant genes are expressed in mouse preimplantation embryos and unfertilized eggs. These mRNA transcripts were detected as maternal mRNA in unfertilized eggs that could delay the lethality of null mutants. All of the proteins were detected in the cytoplasm or in the cell membrane. This study of spatial and temporal expression revealed that all of these six null mutants post-implantation genes in MAPK pathway are expressed and, where tested, phosphorylated/activated proteins are detected in the blastocyst. Studies on RNA expression using RT-PCR suggest that maternal RNA could play an important role in delaying the presence of the lethal phenotype of null mutations. Copyright (c) 2005 Wiley-Liss, Inc.

Analysis of gene expression and Ig transcription in PU.1/Spi-B-deficient progenitor B cell lines.

PubMed

Schweitzer, Brock L; DeKoter, Rodney P

2004-01-01

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1(-/-)Spi-B(-/-) IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1(-/-)Spi-B(-/-) pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igkappa transcription are induced after IL-7 withdrawal of wild-type or PU.1(-/-)Spi-B(-/-) pro-B cells. In contrast, we found that Iglambda transcription is reduced in PU.1(-/-)Spi-B(-/-) pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Iglambda, but not IgH or Igkappa, transcription, is dependent on PU.1 and/or Spi-B. The PU.1(-/-)Spi-B(-/-) pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses

PubMed Central

Vora, Kalpit A; Porter, Gene; Peng, Roche; Cui, Yan; Pryor, Kellyann; Eiermann, George; Zaller, Dennis M

2009-01-01

Background Current literature suggests that dipeptidyl peptidase IV (DPP-IV; CD26) plays an essential role in T-dependent immune responses, a role that could have important clinical consequences. To rigorously define the role of DPP-IV in the immune system, we evaluated genetic and pharmacological inhibition of the enzyme on T-dependent immune responses in vivo. Results The DPP-IV null animals mounted robust primary and secondary antibody responses to the T dependent antigens, 4-hydroxy-3-nitrophenylacetyl-ovalbumin (NP-Ova) and 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin (NP-CGG), which were comparable to wild type mice. Serum levels of antigen specific IgM, IgG1, IgG2a, IgG2b and IgG3 were similar between the two groups of animals. DPP-IV null animals mounted an efficient germinal center reaction by day 10 after antigen stimulation that was comparable to wild type mice. Moreover, the antibodies produced by DPP-IV null animals after repeated antigenic challenge were affinity matured. Similar observations were made using wild type animals treated with a highly selective DPP-IV inhibitor during the entire course of the experiments. T cell recall responses to ovalbumin and MOG peptide, evaluated by measuring proliferation and IL-2 release from cells isolated from draining lymph nodes, were equivalent in DPP-IV null and wild type animals. Furthermore, mice treated with DPP-IV inhibitor had intact T-cell recall responses to MOG peptide. In addition, female DPP-IV null and wild type mice treated with DPP-IV inhibitor exhibited normal and robust in vivo cytotoxic T cell responses after challenge with cells expressing the male H-Y minor histocompatibility antigen. Conclusion These data indicate Selective inhibition of DPP-IV does not impair T dependent immune responses to antigenic challenge. PMID:19358731

Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718.

PubMed

Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A

2011-12-01

Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

Absence of Wip1 partially rescues Atm deficiency phenotypes in mice

PubMed Central

Darlington, Yolanda; Nguyen, Thuy-Ai; Moon, Sung-Hwan; Herron, Alan; Rao, Pulivarthi; Zhu, Chengming; Lu, Xiongbin; Donehower, Lawrence A.

2011-01-01

Wildtype p53-Induced Phosphatase 1 (WIP1) is a serine/threonine phosphatase that dephosphorylates proteins in the ataxia telangiectasia mutated (ATM)-initiated DNA damage response pathway. WIP1 may play a homeostatic role in ATM signaling by returning the cell to a normal pre-stress state following completion of DNA repair. To better understand the effects of WIP1 on ATM signaling, we crossed Atm-deficient mice to Wip1-deficient mice and characterized phenotypes of the double knockout progeny. We hypothesized that the absence of Wip1 might rescue Atm deficiency phenotypes. Atm null mice, like ATM-deficient humans with the inherited syndrome ataxia telangiectasia, exhibit radiation sensitivity, fertility defects, and are T-cell lymphoma prone. Most double knockout mice were largely protected from lymphoma development and had a greatly extended lifespan compared to Atm null mice. Double knockout mice had increased p53 and H2AX phosphorylation and p21 expression compared to their Atm null counterparts, indicating enhanced p53 and DNA damage responses. Additionally, double knockout splenocytes displayed reduced chromosomal instability compared to Atm null mice. Finally, doubly null mice were partially rescued from infertility defects observed in Atm null mice. These results indicate that inhibition of WIP1 may represent a useful strategy for cancer treatment in general and A-T patients in particular. PMID:21765465

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

PubMed

de la Ballina, Laura R; Cano-Crespo, Sara; González-Muñoz, Elena; Bial, Susanna; Estrach, Soline; Cailleteau, Laurence; Tissot, Floriane; Daniel, Hannelore; Zorzano, Antonio; Ginsberg, Mark H; Palacín, Manuel; Féral, Chloé C

2016-04-29

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with β-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Wolfram syndrome 1 gene (WFS1) product localizes to secretory granules and determines granule acidification in pancreatic beta-cells.

PubMed

Hatanaka, Masayuki; Tanabe, Katsuya; Yanai, Akie; Ohta, Yasuharu; Kondo, Manabu; Akiyama, Masaru; Shinoda, Koh; Oka, Yoshitomo; Tanizawa, Yukio

2011-04-01

Wolfram syndrome is an autosomal recessive disorder characterized by juvenile-onset insulin-dependent diabetes mellitus and optic atrophy. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER) resident transmembrane protein. The Wfs1-null mouse exhibits progressive insulin deficiency causing diabetes. Previous work suggested that the function of the WFS1 protein is connected to unfolded protein response and to intracellular Ca(2+) homeostasis. However, its precise molecular function in pancreatic β-cells remains elusive. In our present study, immunofluorescent and electron-microscopic analyses revealed that WFS1 localizes not only to ER but also to secretory granules in pancreatic β-cells. Intragranular acidification was assessed by measuring intracellular fluorescence intensity raised by the acidotrophic agent, 3-[2,4-dinitroanilino]-3'-amino-N-methyldipropyramine. Compared with wild-type β-cells, there was a 32% reduction in the intensity in WFS1-deficient β-cells, indicating the impairment of granular acidification. This phenotype may, at least partly, account for the evidence that Wfs1-null islets have impaired proinsulin processing, resulting in an increased circulating proinsulin level. Morphometric analysis using electron microscopy evidenced that the density of secretory granules attached to the plasma membrane was significantly reduced in Wfs1-null β-cells relative to that in wild-type β-cells. This may be relevant to the recent finding that granular acidification is required for the priming of secretory granules preceding exocytosis and may partly explain the fact that glucose-induced insulin secretion is profoundly impaired in young prediabetic Wfs1-null mice. These results thus provide new insights into the molecular mechanisms of β-cell dysfunction in patients with Wolfram syndrome.

Alfvén wave dynamics at the neighborhood of a 2.5D magnetic null-point

NASA Astrophysics Data System (ADS)

Sabri, S.; Vasheghani Farahani, S.; Ebadi, H.; Hosseinpour, M.; Fazel, Z.

2018-05-01

The aim of the present study is to highlight the energy transfer via the interaction of magnetohydrodynamic waves with a 2.5D magnetic null-point in a finite plasma-β regime of the solar corona. An initially symmetric Alfvén pulse at a specific distance from a magnetic null-point is kicked towards the isothermal null-point. A shock-capturing Godunov-type PLUTO code is used to solve the ideal magnetohydrodynamic set equations in the context of wave-plasma energy transfer. As the Alfvén wave propagates towards the magnetic null-point it experiences speed lowering which ends up in releasing energy along the separatrices. In this line owing to the Alfvén wave, a series of events take place that contribute towards coronal heating. Nonlinear induced waves are by products of the torsional Alfvén interaction with magnetic null-points. The energy of these induced waves which are fast magnetoacoustic (transverse) and slow magnetoacoustic (longitudinal) waves are supplied by the Alfvén wave. The nonlinearly induced density perturbations are proportional to the Alfvén wave energy loss. This supplies energy for the propagation of fast and slow magnetoacoustic waves, where in contrast to the fast wave the slow wave experiences a continuous energy increase. As such, the slow wave may transfer its energy to the medium at later times, maintaining a continuous heating mechanism at the neighborhood of a magnetic null-point.

HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

PubMed

Gavioli, R; Zhang, Q J; Masucci, M G

1996-08-01

The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3.

PubMed

Li, Zhipeng; Li, Xu; Yu, Chao; Wang, Min; Peng, Feng; Xiao, Jie; Tian, Rui; Jiang, Jianxin; Sun, Chengyi

2014-12-01

We intended to investigate the role of microRNA 100 (miR-100) in regulating pancreatic cancer cells' growth in vitro and tumor development in vivo. QTR-PCR was used to examine the expression of miR-100 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-100 mimics (lv-miR-100) was used to overexpress miR-100 in MIA PaCa-2 and FCPAC-1 cells. The effects of overexpressing miR-100 on pancreatic cancer cell proliferation and chemosensitivity to cisplatin were examined by cell proliferation essay in vitro. MIA PaCa-2 cells with endogenously overexpressed miR-100 were transplanted into null mice to examine tumor growth in vivo. The predicted target of miR-100, fibroblast growth factor receptor 3 (FGFR3), was downregulated by siRNA to examine its effect on pancreatic cancer cells. We found miR-100 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lv-miR-100 was able to upregulate endogenous expression of miR-100, inhibited cancer cell proliferation, and increased sensitivities to cisplatin. Overexpressing miR-100 led to significant inhibition on tumor formation in vivo. Luciferase essay showed FGFR3 was direct target of miR-100. FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells and knocking down FGFR3 by siRNA exerted similar effect as miR-100. Our study demonstrated that miR-100 played an important role in pancreatic cancer development, possibly through targeting FGFR3. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

microRNA-137 modulates pancreatic cancer cells tumor growth, invasion and sensitivity to chemotherapy

PubMed Central

Xiao, Jie; Peng, Feng; Yu, Chao; Wang, Min; Li, Xu; Li, Zhipeng; Jiang, Jianxin; Sun, Chengyi

2014-01-01

Background: We intended to investigate the role of microRNA 137 (miR-137) in regulating pancreatic cancer cells’ growth in vitro and tumor development in vivo. Methods: QTR-PCR was used to examine the expression of miR-137 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-137 mimic was used to overexpress miR-137 in PANC-1 and MIA PaCa-2 cells. The effects of overexpressing miR-137 on pancreatic cancer cell invasion and chemo-sensitivity to 5-fluorouracil (5-FU) were examined by cell migration and survival essays in vitro. The molecular target of miR-137, pleiotropic growth factor (PTN), was down-regulated by siRNA to examine its effects on cancer cell invasion. MIA PaCa-2 cells with endogenously overexpressed miR-137 were transplanted into null mice to examine tumor growth in vivo. Results: We found miR-137 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lentivirus containing miR-137 mimic was able to markedly upregulate endogenous expression of miR-137, inhibited cancer cell invasion and increased sensitivities to chemotherapy reagent 5-FU. PTN was significantly down-regulated by overexpressing miR-137 in pancreatic cancer cells, and knocking down PTN was effective to rescue the reduced cancer cell invasion ability caused by miR-137 overexpression. More importantly, overexpressing miR-137 led to significant inhibition on tumor formation, including reductions in tumor weight and tumor size in vivo. Conclusion: Our study demonstrated that miR-137 played an important role in pancreatic cancer development. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer. PMID:25550779

Reduced wheel running and blunted effects of voluntary exercise in LPA1-null mice: the importance of assessing the amount of running in transgenic mice studies.

PubMed

Castilla-Ortega, Estela; Rosell-Valle, Cristina; Blanco, Eduardo; Pedraza, Carmen; Chun, Jerold; Rodríguez de Fonseca, Fernando; Estivill-Torrús, Guillermo; Santín, Luis J

2013-11-01

This work was aimed to assess whether voluntary exercise rescued behavioral and hippocampal alterations in mice lacking the lysophosphatidic acid LPA1 receptor (LPA1-null mice), studying the potential relationship between the amount of exercise performed and its effects. Normal and LPA1-null mice underwent 23 days of free wheel running and were tested for open-field behavior and adult hippocampal neurogenesis (cell proliferation, immature neurons, cell survival). Running decreased anxiety-like behavior in both genotypes but increased exploration only in the normal mice. While running affected all neurogenesis-related measures in normal mice (especially in the suprapyramidal blade of the dentate gyrus), only a moderate increase in cell survival was found in the mutants. Importantly, the LPA1-nulls showed notably reduced running. Analysis suggested that defective running in the LPA1-null mice could contribute to explain the scarce benefit of the voluntary exercise treatment. On the other hand, a literature review revealed that voluntary exercise is frequently used to modulate behavior and the hippocampus in transgenic mice, but half of the studies did not assess the quantity of running, overlooking any potential running impairments. This study adds evidence to the relevance of the quantity of exercise performed, emphasizing the importance of its assessment in transgenic mice research. Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

The role of oxygen as a regulator of stem cell fate during fracture repair in TSP2-null mice.

PubMed

Burke, Darren; Dishowitz, Michael; Sweetwyne, Mariya; Miedel, Emily; Hankenson, Kurt D; Kelly, Daniel J

2013-10-01

It is often difficult to decouple the relative importance of different factors in regulating MSC differentiation. Genetically modified mice provide model systems whereby some variables can be manipulated while others are kept constant. Fracture repair in thrombospondin-2 (TSP2)-null mice is characterized by reduced endochondral ossification and enhanced intramembranous bone formation. The proposed mechanism for this shift in MSC fate is that increased vascular density and hence oxygen availability in TSP2-null mice regulates differentiation. However, TSP2 is multifunctional and regulates other aspects of the regenerative cascade, such as MSC proliferation. The objective of this study is to use a previously developed computational model of tissue differentiation, in which substrate stiffness and oxygen tension regulate stem cell differentiation, to simulate potential mechanisms which may drive alterations in MSC fate in TSP2-null mice. Four models (increased cell proliferation, increased numbers of MSCs in the marrow decreased cellular oxygen consumption, and an initially stiffer callus) were not predictive of experimental observations in TSP2-null mice. In contrast, increasing the rate of angiogenic progression led to a prediction of greater intramembranous ossification, diminished endochondral ossification, and a reduced region of hypoxia in the fracture callus similar to that quantified experimentally by the immunohistochemical detection of pimonidazole adducts that develop with hypoxia. This study therefore provides further support for the hypothesis that oxygen availability during early fracture healing is a key regulator of MSC bipotential differentiation, and furthermore, it highlights the advantages of integrating computational models with genetically modified mouse studies for further elucidating mechanisms regulating stem cell fate. Copyright © 2013 Orthopaedic Research Society.

Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

PubMed

Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

2016-07-11

The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress

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

Cuneo, Kyle C., E-mail: kcuneo@umich.edu; Morgan, Meredith A.; Davis, Mary A.

2016-06-01

Purpose: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G{sub 1} checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). Methods and Materials: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3more » antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. Results: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G{sub 2} arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. Conclusions: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.« less

Whistler mode refraction in highly nonuniform magnetic fields

NASA Astrophysics Data System (ADS)

Urrutia, J. M.; Stenzel, R.

2016-12-01

In a large laboratory plasma the propagation of whistler modes is measured in highly nonuniform magnetic fields created by a current-carrying wires. Ray tracing is not applicable since the wavelength and gradient scale length are comparable. The waves are excited with a loop antenna near the wire. The antenna launches an m=1 helicon mode in a uniform plasma. The total magnetic field consists of a weak uniform background field and a nearly circular field of a straight wire across the background field. A circular loop produces 3D null points and a 2D null line. The whistler wave propagation will be shown. It is relevant to whistler mode propagation in space plasmas near magnetic null-points, small flux ropes, lunar crustal magnetic fields and active wave injection experiments.

The story of DNase II: a stifled death-wish leads to self-harm.

PubMed

Crow, Yanick J

2010-09-01

DNase II is an endonuclease which plays a fundamental role in the degradation of DNA from both apoptotic cells, and nuclei extruded from red blood cells during erythropoiesis: important tasks, considering that everyday 10(8)-10(9) cells undergo apoptosis, and 10(11) red blood cells are produced in the adult human. The DNase II-null mouse demonstrates embryonic lethality due to type I interferon-mediated erythroid precursor cell death triggered by undegraded nucleic acids. However, the mechanisms leading to such cytotoxicity are poorly understood. A study in the current issue of the European Journal of Immunology investigates the role of the death ligand TRAIL in this process. Although TRAIL is shown to be dispensable for the interferon-induced apoptosis of erythroid cells in DNAse II(-/-) embryos, the authors have developed a useful strategy for further exploring this question in future studies. Interestingly, earlier studies by the same group showed that crossing the DNase II-null mouse with a mouse deficient for the type I interferon receptor can rescue the lethal anaemia observed in the DNase II-null embryos, but only at the cost of developing autoimmunity.

Wound Healing Is Defective in Mice Lacking Tetraspanin CD151

PubMed Central

Cowin, Allison J.; Adams, Damian; Geary, Sean M.; Wright, Mark D.; Jones, Jonathan C.R.; Ashman, Leonie K.

2010-01-01

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins α6 β4, α3 β1, and α6 β1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of α6 and β4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on α3 and β1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration. PMID:16410781

Adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter for colorectal cancer.

PubMed

Wang, Zhao-Xia; Bian, Hai-Bo; Yang, Jing-Song; De, Wei; Ji, Xiao-Hui

2009-08-01

Colorectal cancer is a most frequent type of gastrointestinal tract cancers. The prognosis of patients with colorectal cancer remains poor despite intensive interventions. Tumor specific promoter-directed gene therapy and adenoviral technology can be promising strategies for such advanced disease. This study was conducted to explore the possible therapeutic approach of Cox-2 promoter-directed suicide gene therapy with herpes simplex virus thymidine kinase (HSV-tk) in combination with adenoviral technology for advanced colorectal cancer. Firstly, the activity of Cox-2 promoter was assessed by dual luciferase and enhanced green fluorescent protein reporter gene assays in colorectal cancer cell lines and normal human intestinal epithelial cell line. Then, the expression of coxsackievirus and adenovirus receptor (CAR) was detected in colorectal cancer cell lines. The Cox-2 promoter-directed HSV-tk/ganciclovir (GCV) system mediated by adenovirus (Ad-Cp-TK) was developed (Ad-CMVp-TK, Ad-null and no Ad as controls). In vitro cytoxicity, colony formation and apoptosis assays were performed using Ad-Cp-TK. An animal study was carried out in which BALB/C nude mice bearing tumors were treated with Ad-Cp-TK and GCV treatments. Results showed that Cox-2 promoter possessed high transcriptional activity in a tumor-specific manner. All colorectal cancer cells were detected CAR-positive. In vitro cytotoxic and colony formation assays showed that colorectal cancer cells infected with Ad-Cp-TK became more sensitive to GCV but the sensitivity of normal cells infected with Ad-Cp-TK to GCV were not altered. Moreover, the Ad-Cp-TK system combined with GCV treatment could significantly induce apoptosis of colorectal cancer cells but not normal intestinal epithelial cells. Furthermore, this system also significantly inhibited the growth of subcutaneous tumors and prolonged survival of mice. Thus, adenovirus primary receptor was positive in colorectal cancer cells and adenovirus-mediated suicide gene therapy under the control of Cox-2 promoter could provide a promising treatment modality for advanced colorectal cancer with tumor specificity.

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells.

PubMed

Rathos, Maggie J; Khanwalkar, Harshal; Joshi, Kavita; Manohar, Sonal M; Joshi, Kalpana S

2013-01-23

In the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells. Synergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model. The combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model. These findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression.

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells

PubMed Central

2013-01-01

Background In the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells. Methods Synergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model. Results The combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model. Conclusion These findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression. PMID:23343191

A ‘synthetic-sickness’ screen for senescence re-engagement targets in mutant cancer backgrounds

PubMed Central

Godwin, Lauren S.; Bilsland, Alan E.; Stevenson, Katrina H.; Moore, Jon D.; Wiggins, Ceri M.; Collinson, Rebecca S.; Mudd, Clare; Sadaie, Mahito; Bennett, Dorothy C.; Torrance, Christopher J.; Keith, W. Nicol

2017-01-01

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments. PMID:28806777

The GDNF Target Vsnl1 Marks the Ureteric Tip

PubMed Central

Ola, Roxana; Jakobson, Madis; Kvist, Jouni; Perälä, Nina; Kuure, Satu; Braunewell, Karl-Heinz; Bridgewater, Darren; Rosenblum, Norman D.; Chilov, Dmitri; Immonen, Tiina; Sainio, Kirsi

2011-01-01

Glial cell line-derived neurotrophic factor (GDNF) is indispensable for ureteric budding and branching. If applied exogenously, GDNF promotes ectopic ureteric buds from the Wolffian duct. Although several downstream effectors of GDNF are known, the identification of early response genes is incomplete. Here, microarray screening detected several GDNF-regulated genes in the Wolffian duct, including Visinin like 1 (Vsnl1), which encodes a neuronal calcium-sensor protein. We observed renal Vsnl1 expression exclusively in the ureteric epithelium, but not in Gdnf-null kidneys. In the tissue culture of Gdnf-deficient kidney primordium, exogenous GDNF and alternative bud inducers (FGF7 and follistatin) restored Vsnl1 expression. Hence, Vsnl1 characterizes the tip of the ureteric bud epithelium regardless of the inducer. In the tips, Vsnl1 showed a mosaic expression pattern that was mutually exclusive with β-catenin transcriptional activation. Vsnl1 was downregulated in both β-catenin-stabilized and β-catenin-deficient kidneys. Moreover, in a mouse collecting duct cell line, Vsnl1 compromised β-catenin stability, suggesting a counteracting relationship between Vsnl1 and β-catenin. In summary, Vsnl1 marks ureteric bud tips in embryonic kidneys, and its mosaic pattern demonstrates a heterogeneity of cell types that may be critical for normal ureteric branching. PMID:21289216

FOXA1 is an essential determinant of ERα expression and mammary ductal morphogenesis

PubMed Central

Bernardo, Gina M.; Lozada, Kristen L.; Miedler, John D.; Harburg, Gwyndolen; Hewitt, Sylvia C.; Mosley, Jonathan D.; Godwin, Andrew K.; Korach, Kenneth S.; Visvader, Jane E.; Kaestner, Klaus H.; Abdul-Karim, Fadi W.; Montano, Monica M.; Keri, Ruth A.

2010-01-01

FOXA1, estrogen receptor α (ERα) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERα and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERα expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERα and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERα expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERα-positive breast cancers, at least in part, through its control of ERα expression. PMID:20501593

Role of RANKL (TNFSF11)-Dependent Osteopetrosis in the Dental Phenotype of Msx2 Null Mutant Mice

PubMed Central

Castaneda, Beatriz; Simon, Yohann; Ferbus, Didier; Robert, Benoit; Chesneau, Julie; Mueller, Christopher

2013-01-01

The MSX2 homeoprotein is implicated in all aspects of craniofacial skeletal development. During postnatal growth, MSX2 is expressed in all cells involved in mineralized tissue formation and plays a role in their differentiation and function. Msx2 null (Msx2 −/−) mice display complex craniofacial skeleton abnormalities with bone and tooth defects. A moderate form osteopetrotic phenotype is observed, along with decreased expression of RANKL (TNFSF11), the main osteoclast-differentiating factor. In order to elucidate the role of such an osteopetrosis in the Msx2 −/− mouse dental phenotype, a bone resorption rescue was performed by mating Msx2 −/− mice with a transgenic mouse line overexpressing Rank (Tnfrsf11a). Msx2 −/− RankTg mice had significant improvement in the molar phenotype, while incisor epithelium defects were exacerbated in the enamel area, with formation of massive osteolytic tumors. Although compensation for RANKL loss of function could have potential as a therapy for osteopetrosis, but in Msx2 −/− mice, this approach via RANK overexpression in monocyte-derived lineages, amplified latent epithelial tumor development in the peculiar continuously growing incisor. PMID:24278237

Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia.

PubMed

Manfroi, Silvia; Scarcello, Antonio; Pagliaro, Pasqualepaolo

2015-10-01

Molecular genetic studies on Duffy blood group antigens have identified mutations underlying rare FY*Null and FY*X alleles. FY*Null has a high frequency in Blacks, especially from sub-Saharan Africa, while its frequency is not defined in Caucasians. FY*X allele, associated with Fy(a-b+w) phenotype, has a frequency of 2-3.5% in Caucasian people while it is absent in Blacks. During the project of extensive blood group genotyping in patients affected by hemoglobinopathies, we identified FY*X/FY*Null and FY*A/FY*Null genotypes in a Caucasian thalassemic family from Sardinia. We speculate on the frequency of FY*X and FY*Null alleles in Caucasian and Black people; further, we focused on the association of FY*X allele with weak Fyb antigen expression on red blood cells and its identification performing high sensitivity serological typing methods or genotyping. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of null-point detection methods on simulation data

NASA Astrophysics Data System (ADS)

Olshevsky, Vyacheslav; Fu, Huishan; Vaivads, Andris; Khotyaintsev, Yuri; Lapenta, Giovanni; Markidis, Stefano

2014-05-01

We model the measurements of artificial spacecraft that resemble the configuration of CLUSTER propagating in the particle-in-cell simulation of turbulent magnetic reconnection. The simulation domain contains multiple isolated X-type null-points, but the majority are O-type null-points. Simulations show that current pinches surrounded by twisted fields, analogous to laboratory pinches, are formed along the sequences of O-type nulls. In the simulation, the magnetic reconnection is mainly driven by the kinking of the pinches, at spatial scales of several ion inertial lentghs. We compute the locations of magnetic null-points and detect their type. When the satellites are separated by the fractions of ion inertial length, as it is for CLUSTER, they are able to locate both the isolated null-points, and the pinches. We apply the method to the real CLUSTER data and speculate how common are pinches in the magnetosphere, and whether they play a dominant role in the dissipation of magnetic energy.

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

PubMed Central

Lo, Pang-Kuo; Lee, Ji Shin; Sukumar, Saraswati

2011-01-01

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G1 arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G1 arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1. PMID:21964066

Suppression of AKT phosphorylation restores rapamycin-based synthetic lethality in SMAD4-defective pancreatic cancer cells.

PubMed

Le Gendre, Onica; Sookdeo, Ayisha; Duliepre, Stephie-Anne; Utter, Matthew; Frias, Maria; Foster, David A

2013-05-01

mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers-with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. ©2013 AACR.

Guttiferone K impedes cell cycle re-entry of quiescent prostate cancer cells via stabilization of FBXW7 and subsequent c-MYC degradation.

PubMed

Xi, Z; Yao, M; Li, Y; Xie, C; Holst, J; Liu, T; Cai, S; Lao, Y; Tan, H; Xu, H-X; Dong, Q

2016-06-02

Cell cycle re-entry by quiescent cancer cells is an important mechanism for cancer progression. While high levels of c-MYC expression are sufficient for cell cycle re-entry, the modality to block c-MYC expression, and subsequent cell cycle re-entry, is limited. Using reversible quiescence rendered by serum withdrawal or contact inhibition in PTEN(null)/p53(WT) (LNCaP) or PTEN(null)/p53(mut) (PC-3) prostate cancer cells, we have identified a compound that is able to impede cell cycle re-entry through c-MYC. Guttiferone K (GUTK) blocked resumption of DNA synthesis and preserved the cell cycle phase characteristics of quiescent cells after release from the quiescence. In vehicle-treated cells, there was a rapid increase in c-MYC protein levels upon release from the quiescence. However, this increase was inhibited in the presence of GUTK with an associated acceleration in c-MYC protein degradation. The inhibitory effect of GUTK on cell cycle re-entry was significantly reduced in cells overexpressing c-MYC. The protein level of FBXW7, a subunit of E3 ubiquitin ligase responsible for degradation of c-MYC, was reduced upon the release from the quiescence. In contrast, GUTK stabilized FBXW7 protein levels during release from the quiescence. The critical role of FBXW7 was confirmed using siRNA knockdown, which impaired the inhibitory effect of GUTK on c-MYC protein levels and cell cycle re-entry. Administration of GUTK, either in vitro prior to transplantation or in vivo, suppressed the growth of quiescent prostate cancer cell xenografts. Furthermore, elevation of FBXW7 protein levels and reduction of c-MYC protein levels were found in the xenografts of GUTK-treated compared with vehicle-treated mice. Hence, we have identified a compound that is capable of impeding cell cycle re-entry by quiescent PTEN(null)/p53(WT) and PTEN(null)/p53(mut) prostate cancer cells likely by promoting c-MYC protein degradation through stabilization of FBXW7. Its usage as a clinical modality to prevent prostate cancer progression should be further evaluated.

Regulation of D-cyclin translation inhibition in myeloma cells treated with mTOR inhibitors: Rationale for combined treatment with ERK inhibitors and rapamycin

PubMed Central

Frost, Patrick; Shi, Yijiang; Hoang, Bao; Gera, Joseph; Lichtenstein, Alan

2009-01-01

We have shown that heightened AKT activity sensitized multiple myeloma (MM) cells to the anti-tumor effects of the mTOR-inhibitor, CCI-779. To test the mechanism of AKT’s regulatory role, we stably transfected U266 MM cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analog, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mTOR inhibitors to downregulate D-cyclin expression was significantly greater in AKT-transfected MM cells, due in part, to AKT’s ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was demonstrated in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild type PTEN. As ERK/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells demonstrated significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. However, ectopic over-expression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high AKT” myeloma cells indicating that MEK/ERK activity was required but not sufficient for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in MM cells and ERK facilitates activity. PMID:19139116

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

PubMed Central

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H.; Mills, Jason A.; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M.; Podsakoff, Gregory M.; Gadue, Paul; French, Deborah L.; Mason, Philip J.; Bessler, Monica

2013-01-01

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the “safe harbor” AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells. PMID:23744582

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

PubMed

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H; Mills, Jason A; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M; Podsakoff, Gregory M; Gadue, Paul; French, Deborah L; Mason, Philip J; Bessler, Monica; Weiss, Mitchell J

2013-08-08

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Syndecan-4 shedding impairs macrovascular angiogenesis in diabetes mellitus

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

Li, Ran; Xie, Jun; Wu, Han

Purpose: Syndecan-4 (synd4) is a ubiquitous heparan sulfate proteoglycan cell surface receptor that modulates cell proliferation, migration, mechanotransduction, and endocytosis. The extracellular domain of synd4 sheds heavily in acute inflammation, but the shedding of synd4 in chronic inflammation, such as diabetes mellitus (DM), is still undefined. We investigated the alterations of synd4 endothelial expression in DM and the influence of impaired synd4 signaling on angiogenesis in human umbilical vein endothelial cells (HUVECs), diabetic rats, synd4 null mice, and db/db mice. Material and methods: HUVECs were incubated with advanced glycation end products (AGEs). Western blot analysis was used to determine synd4more » protein expression and ELISA was used to detect soluble synd4 fragments. The concentration of synd4 in the aortic endothelia of diabetic rats was detected by immunohistochemical staining. Aortic ring assays were performed to study the process of angiogenesis in the diabetic rats and in synd4 null and db/db mice. Recombinant adenoviruses containing the synd4 gene or null were constructed to enhance synd4 aortic expression in db/db mice. Results: Western blot analysis showed decreased expression of the synd4 extracellular domain in HUVECs, and ELISA detected increased soluble fragments of synd4 in the media. Synd4 endothelial expression in the aortas of diabetic rats was decreased. Aortic ring assay indicated impaired angiogenesis in synd4 null and db/db mice, which was partially reversed by synd4 overexpression in db/db mice. Conclusion: Synd4 shedding from vascular endothelial cells played an important role in the diabetes-related impairment of angiogenesis. -- Highlights: •Synd4 shedding from endothelial cells is accelerated under the stimulation of AGEs. •Extracellular domain of synd4 is diminished in the endothelium of DM rats. •Aortic rings of synd4 null mice showed impaired angiogenesis. •Overexpression of synd4 partly rescues macrovascular angiogenesis in db/db mice.« less

Neural cell adhesion molecule, NCAM, regulates thalamocortical axon pathfinding and the organization of the cortical somatosensory representation in mouse

PubMed Central

Enriquez-Barreto, Lilian; Palazzetti, Cecilia; Brennaman, Leann H.; Maness, Patricia F.; Fairén, Alfonso

2012-01-01

To study the potential role of neural cell adhesion molecule (NCAM) in the development of thalamocortical (TC) axon topography, wild type, and NCAM null mutant mice were analyzed for NCAM expression, projection, and targeting of TC afferents within the somatosensory area of the neocortex. Here we report that NCAM and its α-2,8-linked polysialic acid (PSA) are expressed in developing TC axons during projection to the neocortex. Pathfinding of TC axons in wild type and null mutant mice was mapped using anterograde DiI labeling. At embryonic day E16.5, null mutant mice displayed misguided TC axons in the dorsal telencephalon, but not in the ventral telencephalon, an intermediate target that initially sorts TC axons toward correct neocortical areas. During the early postnatal period, rostrolateral TC axons within the internal capsule along the ventral telencephalon adopted distorted trajectories in the ventral telencephalon and failed to reach the neocortex in NCAM null mutant animals. NCAM null mutants showed abnormal segregation of layer IV barrels in a restricted portion of the somatosensory cortex. As shown by Nissl and cytochrome oxidase staining, barrels of the anterolateral barrel subfield (ALBSF) and the most distal barrels of the posteromedial barrel subfield (PMBSF) did not segregate properly in null mutant mice. These results indicate a novel role for NCAM in axonal pathfinding and topographic sorting of TC axons, which may be important for the function of specific territories of sensory representation in the somatosensory cortex. PMID:22723769

Alterations in Corneal Sensory Nerves During Homeostasis, Aging, and After Injury in Mice Lacking the Heparan Sulfate Proteoglycan Syndecan-1.

PubMed

Pal-Ghosh, Sonali; Tadvalkar, Gauri; Stepp, Mary Ann

2017-10-01

To determine the impact of the loss of syndecan 1 (SDC1) on intraepithelial corneal nerves (ICNs) during homeostasis, aging, and in response to 1.5-mm trephine and debridement injury. Whole-mount corneas are used to quantify ICN density and thickness over time after birth and in response to injury in SDC1-null and wild-type (WT) mice. High-resolution three-dimensional imaging is used to visualize intraepithelial nerve terminals (INTs), axon fragments, and lysosomes in corneal epithelial cells using antibodies against growth associated protein 43 (GAP43), βIII tubulin, and LAMP1. Quantitative PCR was performed to quantify expression of SDC1, SDC2, SDC3, and SDC4 in corneal epithelial mRNA. Phagocytosis was assessed by quantifying internalization of fluorescently labeled 1-μm latex beads. Intraepithelial corneal nerves innervate the corneas of SDC1-null mice more slowly. At 8 weeks, ICN density is less but thickness is greater. Apically projecting intraepithelial nerve terminals and lysosome-associated membrane glycoprotein 1 (LAMP1) are also reduced in unwounded SDC1-null corneas. Quantitative PCR and immunofluorescence studies show that SDC3 expression and localization are increased in SDC1-null ICNs. Wild-type and SDC1-null corneas lose ICN density and thickness as they age. Recovery of axon density and thickness after trephine but not debridement wounds is slower in SDC1-null corneas compared with WT. Experiments assessing phagocytosis show reduced bead internalization by SDC1-null epithelial cells. Syndecan-1 deficiency alters ICN morphology and homeostasis during aging, reduces epithelial phagocytosis, and impairs reinnervation after trephine but not debridement injury. These data provide insight into the mechanisms used by sensory nerves to reinnervate after injury.

Bit-1 is an essential regulator of myogenic differentiation

PubMed Central

Griffiths, Genevieve S.; Doe, Jinger; Jijiwa, Mayumi; Van Ry, Pam; Cruz, Vivian; de la Vega, Michelle; Ramos, Joe W.; Burkin, Dean J.; Matter, Michelle L.

2015-01-01

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2. PMID:25770104

Impaired olfaction in mice lacking aquaporin-4 water channels.

PubMed

Lu, Daniel C; Zhang, Hua; Zador, Zsolt; Verkman, A S

2008-09-01

Aquaporin-4 (AQP4) is a water-selective transport protein expressed in glial cells throughout the central nervous system. AQP4 deletion in mice produces alterations in several neuroexcitation phenomena, including hearing, vision, epilepsy, and cortical spreading depression. Here, we report defective olfaction and electroolfactogram responses in AQP4-null mice. Immunofluorescence indicated strong AQP4 expression in supportive cells of the nasal olfactory epithelium. The olfactory epithelium in AQP4-null mice had identical appearance, but did not express AQP4, and had approximately 12-fold reduced osmotic water permeability. Behavioral analysis showed greatly impaired olfaction in AQP4-null mice, with latency times of 17 +/- 0.7 vs. 55 +/- 5 s in wild-type vs. AQP4-null mice in a buried food pellet test, which was confirmed using an olfactory maze test. Electroolfactogram voltage responses to multiple odorants were reduced in AQP4-null mice, with maximal responses to triethylamine of 0.80 +/- 0.07 vs. 0.28 +/- 0.03 mV. Similar olfaction and electroolfactogram defects were found in outbred (CD1) and inbred (C57/bl6) mouse genetic backgrounds. Our results establish AQP4 as a novel determinant of olfaction, the deficiency of which probably impairs extracellular space K(+) buffering in the olfactory epithelium.

ZBTB20 is required for anterior pituitary development and lactotrope specification.

PubMed

Cao, Dongmei; Ma, Xianhua; Cai, Jiao; Luan, Jing; Liu, An-Jun; Yang, Rui; Cao, Yi; Zhu, Xiaotong; Zhang, Hai; Chen, Yu-Xia; Shi, Yuguang; Shi, Guang-Xia; Zou, Dajin; Cao, Xuetao; Grusby, Michael J; Xie, Zhifang; Zhang, Weiping J

2016-04-15

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

ZBTB20 is required for anterior pituitary development and lactotrope specification

PubMed Central

Cao, Dongmei; Ma, Xianhua; Cai, Jiao; Luan, Jing; Liu, An-Jun; Yang, Rui; Cao, Yi; Zhu, Xiaotong; Zhang, Hai; Chen, Yu-Xia; Shi, Yuguang; Shi, Guang-Xia; Zou, Dajin; Cao, Xuetao; Grusby, Michael J.; Xie, Zhifang; Zhang, Weiping J.

2016-01-01

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification. PMID:27079169

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status

PubMed Central

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G.; Wong, Kwok-Kin; Khuri, Fadlo R.; Zhou, Wei; Shin, Dong M.

2017-01-01

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 (krasG12D/wt/p53-/-/lkb1wt/wt) and t2 (krasG12D/wt/p53-/-/lkb1-/-) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer. PMID:28938614

The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine

PubMed Central

Walsby, Elisabeth J.; Coles, Steven J.; Knapper, Steven; Burnett, Alan K.

2011-01-01

Background Topoisomerase II is essential for the maintenance of DNA integrity and the survival of proliferating cells. Topoisomerase II poisons, including etoposide and doxorubicin, inhibit enzyme-mediated DNA ligation causing the accumulation of double-stranded breaks and have been front-line drugs for the treatment of leukemia for many years. Voreloxin is a first-in-class anti-cancer quinolone derivative that intercalates DNA and inhibits topoisomerase II. The efficacy and mechanisms of action of voreloxin in acute myeloid leukaemia were addressed in this study. Design and Methods Primary acute myeloid leukemia blasts (n = 88) and myeloid cell lines were used in vitro to study voreloxin through viability assays to assess cell killing and synergy with other drugs. Apoptosis and cell cycling were assessed by flow cytometry. DNA relaxation assays were utilized to determine that voreloxin was active on topoisomerase II. Results The mean lethal dose 50% (LD50) (± standard deviation) of voreloxin for primary acute myeloid leukemia blasts was 2.30 μM (± 1.87). Synergy experiments between voreloxin and cytarabine identified synergism in 22 of 25 primary acute myeloid leukemia samples tested, with a mean combination index of 0.79. Apoptosis was shown to increase in a dose-dependent manner. Furthermore, voreloxin was active in the p53-null K562 cell line suggesting that the action of voreloxin is not affected by p53 status. The action of voreloxin on topoisomerase II was confirmed using a DNA relaxation assay. Conclusions Voreloxin may provide an interesting addition to the cache of drugs available for the treatment of acute myeloid leukemia, a disease with a poor long-term survival. In addition to its potent action as a single agent in dividing cells, the synergy we demonstrated between voreloxin and cytarabine recommends further investigation of this topoisomerase II inhibitor. PMID:21134979

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

Olshevsky, Vyacheslav; Lapenta, Giovanni; Divin, Andrey

We use kinetic particle-in-cell and MHD simulations supported by an observational data set to investigate magnetic reconnection in clusters of null points in space plasma. The magnetic configuration under investigation is driven by fast adiabatic flux rope compression that dissipates almost half of the initial magnetic field energy. In this phase powerful currents are excited producing secondary instabilities, and the system is brought into a state of “intermittent turbulence” within a few ion gyro-periods. Reconnection events are distributed all over the simulation domain and energy dissipation is rather volume-filling. Numerous spiral null points interconnected via their spines form null linesmore » embedded into magnetic flux ropes; null point pairs demonstrate the signatures of torsional spine reconnection. However, energy dissipation mainly happens in the shear layers formed by adjacent flux ropes with oppositely directed currents. In these regions radial null pairs are spontaneously emerging and vanishing, associated with electron streams and small-scale current sheets. The number of spiral nulls in the simulation outweighs the number of radial nulls by a factor of 5–10, in accordance with Cluster observations in the Earth's magnetosheath. Twisted magnetic fields with embedded spiral null points might indicate the regions of major energy dissipation for future space missions such as the Magnetospheric Multiscale Mission.« less

Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia

NASA Technical Reports Server (NTRS)

Fritzsch, Bernd

2003-01-01

The molecular and cellular origin of the primary neurons of the inner ear, the vestibular and spiral neurons, is reviewed including how they connect to the specific sensory epithelia and what the molecular nature of their survival is. Primary neurons of the ear depend on a single basic Helix-Loop-Helix (bHLH) protein for their formation, neurogenin 1 (ngn1). An immediate downstream gene is the bHLH gene neuronal differentiation (NeuroD). Targeted null mutations of ngn1 results in absence of primary neuron formation; targeted null mutation of NeuroD results in loss of almost all spiral and many vestibular neurons. NeuroD and a later expressed gene, Brn3a, play a role in pathfinding to and within sensory epithelia. The molecular nature of this pathfinding property is unknown. Reduction of hair cells in ngn1 null mutations suggests a clonal relationship with primary neurons. This relationship may play some role in specifying the identity of hair cells and the primary neurons that connect with them. Primary neuron neurites growth to sensory epithelia is initially independent of trophic factors released from developing sensory epithelia, but becomes rapidly dependent on those factors. Null mutations of specific neurotrophic factors lose distinct primary neuron populations which undergo rapid embryonic cell death.

Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.

PubMed

Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I; Glogauer, Michael; McCulloch, Christopher A; Everts, Vincent

2017-08-01

Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP + osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm 2 ; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.

Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and CytokinesisD⃞V⃞

PubMed Central

Chen, Qian; Li, Hui; De Lozanne, Arturo

2006-01-01

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring. PMID:16339076

Roles of an Unconventional Protein Kinase and Myosin II in Amoeba Osmotic Shock Responses

PubMed Central

Betapudi, Venkaiah; Egelhoff, Thomas T.

2009-01-01

The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. GFP-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical, and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa. PMID:19843280

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.

PubMed

Yang, Zhong-Fa; Zhang, Haojian; Ma, Leyuan; Peng, Cong; Chen, Yaoyu; Wang, Junling; Green, Michael R; Li, Shaoguang; Rosmarin, Alan G

2013-02-05

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

PubMed

Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication.

Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

PubMed

Wiegering, Armin; Matthes, Niels; Mühling, Bettina; Koospal, Monika; Quenzer, Anne; Peter, Stephanie; Germer, Christoph-Thomas; Linnebacher, Michael; Otto, Christoph

2017-04-01

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC 50 <3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase.

PubMed

Ng, Ho Yin; Oliver, Brian Gregory George; Burgess, Janette Kay; Krymskaya, Vera P; Black, Judith Lee; Moir, Lyn M

2015-11-01

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Interferometric rotation sensor

NASA Technical Reports Server (NTRS)

Walsh, T. M.

1972-01-01

Sensor generates interference fringes varying in number (horizontally and vertically) as a function of the total angular deviation relative to the line-of-sight axis. Device eliminates errors from zero or null shift due to lack of electrical circuitry stability.

Loss of the clock protein PER2 shortens the erythrocyte life span in mice.

PubMed

Sun, Qi; Zhao, Yue; Yang, Yunxia; Yang, Xiao; Li, Minghui; Xu, Xi; Wen, Dan; Wang, Junsong; Zhang, Jianfa

2017-07-28

Cell proliferation and release from the bone marrow have been demonstrated to be controlled by circadian rhythms in both humans and mice. However, it is unclear whether local circadian clocks in the bone marrow influence physiological functions and life span of erythrocytes. Here, we report that loss of the clock gene Per2 significantly decreased erythrocyte life span. Mice deficient in Per2 were more susceptible to acute stresses in the erythrocytes, becoming severely anemic upon phenylhydrazine, osmotic, and H 2 O 2 challenges. 1 H NMR-based metabolomics analysis revealed that the Per2 depletion causes significant changes in metabolic profiles of erythrocytes, including increased lactate and decreased ATP levels compared with wild-type mice. The lower ATP levels were associated with hyperfunction of Na + /K + -ATPase activity in Per2 -null erythrocytes, and inhibition of Na + /K + -ATPase activity by ouabain efficiently rescued ATP levels. Per2 -null mice displayed increased levels of Na + /K + -ATPase α1 (ATP1A1) in the erythrocyte membrane, and transfection of Per2 cDNA into the erythroleukemic cell line TF-1 inhibited Atp1a1 expression. Furthermore, we observed that PER2 regulates Atp1a1 transcription through interacting with trans-acting transcription factor 1 (SP1). Our findings reveal that Per2 function in the bone marrow is required for the regulation of life span in circulating erythrocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans.

PubMed

Mainpal, Rana; Nance, Jeremy; Yanowitz, Judith L

2015-10-15

Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation. © 2015. Published by The Company of Biologists Ltd.

Cytosolic T3-binding protein modulates dynamic alteration of T3-mediated gene expression in cells.

PubMed

Takeshige, Keiko; Sekido, Takashi; Kitahara, Jun-ichirou; Ohkubo, Yousuke; Hiwatashi, Dai; Ishii, Hiroaki; Nishio, Shin-ichi; Takeda, Teiji; Komatsu, Mitsuhisa; Suzuki, Satoru

2014-01-01

μ-Crystallin (CRYM) is also known as NADPH-dependent cytosolic T3-binding protein. A study using CRYM-null mice suggested that CRYM stores triiodothyronine (T3) in tissues. We previously established CRYM-expressing cells derived from parental GH3 cells. To examine the precise regulation of T3-responsive genes in the presence of CRYM, we evaluated serial alterations of T3-responsive gene expression by changing pericellular T3 concentrations in the media. We estimated the constitutive expression of three T3-responsive genes, growth hormone (GH), deiodinase 1 (Dio1), and deiodinase 2 (Dio2), in two cell lines. Subsequently, we measured the responsiveness of these three genes at 4, 8, 16, and 24 h after adding various concentrations of T3. We also estimated the levels of these mRNAs 24 and 48 h after removing T3. The levels of constitutive expression of GH and Dio1 were low and high in C8 cells, respectively, while Dio2 expression was not significantly different between GH3 and C8 cells. When treated with T3, Dio2 expression was significantly enhanced in C8 cells, while there were no differences in GH or Dio1 expression between GH3 and C8 cell lines. In contrast, removal of T3 retained the mRNA expression of GH and Dio2 in C8 cells. These results suggest that CRYM expression increases and sustains the T3 responsiveness of genes in cells, especially with alteration of the pericellular T3 concentration. The heterogeneity of T3-related gene expression is dependent on cellular CRYM expression in cases of dynamic changes in pericellular T3 concentration.

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health

PubMed Central

Suyama, Kaori; Silagi, Elizabeth S.; Choi, Hyowon; Sakabe, Kou; Mochida, Joji; Shapiro, Irving M.; Risbud, Makarand V.

2016-01-01

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche. PMID:27049729

The glutathione-S-transferase mu 1 (GSTM1) null genotype and increased neutrophil response to low-level ozone (0.06 ppm).

EPA Science Inventory

Background: Exposure of healthy young adults to 03 modulates immune cell biology in the airways and causes a significant increase in neutrophilic inflammation which can vary considerably in magnitude across individuals. The GSTM1null genotype modulates Oj-induced inflammation, bu...

Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

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

Sakamoto, Youhei; Hara, Kenshiro; Kanai-Azuma, Masami

Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posteriormore » dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.« less

The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression

PubMed Central

Kroken, Abby R.; Chen, Camille K.; Evans, David J.; Yahr, Timothy L.

2018-01-01

ABSTRACT Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103ΔexoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis. PMID:29717012

Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer-aided simulations.

PubMed

Okahashi, Nobuyuki; Kohno, Susumu; Kitajima, Shunsuke; Matsuda, Fumio; Takahashi, Chiaki; Shimizu, Hiroshi

2015-12-01

Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, (13)C-labeling experiments, and computer-aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53-null mice. Cells were cultured in medium containing [1-(13)C] glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH). The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time-course analysis of cultured media. Gas chromatography-mass spectrometry analysis of the (13)C-labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer-aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53-null mice, and also demonstrated that reductive glutamine metabolism is active in p53-null soft tissue sarcoma cells under normoxia. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.

PubMed

Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

2010-12-28

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

PubMed Central

Omori, Yoshihiro; Chaya, Taro; Katoh, Kimiko; Kajimura, Naoko; Sato, Shigeru; Muraoka, Koichiro; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

2010-01-01

Cilia function as cell sensors in many organs, and their disorders are referred to as “ciliopathies.” Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors. PMID:21148103

Fascin1-Dependent Filopodia are Required for Directional Migration of a Subset of Neural Crest Cells

PubMed Central

Boer, Elena F.; Howell, Elizabeth D.; Schilling, Thomas F.; Jette, Cicely A.; Stewart, Rodney A.

2015-01-01

Directional migration of neural crest (NC) cells is essential for patterning the vertebrate embryo, including the craniofacial skeleton. Extensive filopodial protrusions in NC cells are thought to sense chemo-attractive/repulsive signals that provide directionality. To test this hypothesis, we generated null mutations in zebrafish fascin1a (fscn1a), which encodes an actin-bundling protein required for filopodia formation. Homozygous fscn1a zygotic null mutants have normal NC filopodia due to unexpected stability of maternal Fscn1a protein throughout NC development and into juvenile stages. In contrast, maternal/zygotic fscn1a null mutant embryos (fscn1a MZ) have severe loss of NC filopodia. However, only a subset of NC streams display migration defects, associated with selective loss of craniofacial elements and peripheral neurons. We also show that fscn1a-dependent NC migration functions through cxcr4a/cxcl12b chemokine signaling to ensure the fidelity of directional cell migration. These data show that fscn1a-dependent filopodia are required in a subset of NC cells to promote cell migration and NC derivative formation, and that perdurance of long-lived maternal proteins can mask essential zygotic gene functions during NC development. PMID:25607881

Abnormal Mammary Development in 129:STAT1-Null Mice is Stroma-Dependent

PubMed Central

Cardiff, Robert D.; Trott, Josephine F.; Hovey, Russell C.; Hubbard, Neil E.; Engelberg, Jesse A.; Tepper, Clifford G.; Willis, Brandon J.; Khan, Imran H.; Ravindran, Resmi K.; Chan, Szeman R.; Schreiber, Robert D.; Borowsky, Alexander D.

2015-01-01

Female 129:Stat1-null mice (129S6/SvEvTac-Stat1tm1Rds homozygous) uniquely develop estrogen-receptor (ER)-positive mammary tumors. Herein we report that the mammary glands (MG) of these mice have altered growth and development with abnormal terminal end buds alongside defective branching morphogenesis and ductal elongation. We also find that the 129:Stat1-null mammary fat pad (MFP) fails to sustain the growth of 129S6/SvEv wild-type and Stat1-null epithelium. These abnormalities are partially reversed by elevated serum progesterone and prolactin whereas transplantation of wild-type bone marrow into 129:Stat1-null mice does not reverse the MG developmental defects. Medium conditioned by 129:Stat1-null epithelium-cleared MFP does not stimulate epithelial proliferation, whereas it is stimulated by medium conditioned by epithelium-cleared MFP from either wild-type or 129:Stat1-null females having elevated progesterone and prolactin. Microarrays and multiplexed cytokine assays reveal that the MG of 129:Stat1-null mice has lower levels of growth factors that have been implicated in normal MG growth and development. Transplanted 129:Stat1-null tumors and their isolated cells also grow slower in 129:Stat1-null MG compared to wild-type recipient MG. These studies demonstrate that growth of normal and neoplastic 129:Stat1-null epithelium is dependent on the hormonal milieu and on factors from the mammary stroma such as cytokines. While the individual or combined effects of these factors remains to be resolved, our data supports the role of STAT1 in maintaining a tumor-suppressive MG microenvironment. PMID:26075897

CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.

PubMed

Le Floch, Renaud; Chiche, Johanna; Marchiq, Ibtissam; Naiken, Tanesha; Naïken, Tanesha; Ilc, Karine; Ilk, Karine; Murray, Clare M; Critchlow, Susan E; Roux, Danièle; Simon, Marie-Pierre; Pouysségur, Jacques

2011-10-04

Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.

CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors

PubMed Central

Le Floch, Renaud; Chiche, Johanna; Marchiq, Ibtissam; Naiken, Tanesha; Ilc, Karine; Murray, Clare M.; Critchlow, Susan E.; Roux, Danièle; Simon, Marie-Pierre; Pouysségur, Jacques

2011-01-01

Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H+/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res−) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res− cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H+ symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basiginhigh cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy. PMID:21930917

Nitric oxide negatively regulates mammalian adult neurogenesis

NASA Astrophysics Data System (ADS)

Packer, Michael A.; Stasiv, Yuri; Benraiss, Abdellatif; Chmielnicki, Eva; Grinberg, Alexander; Westphal, Heiner; Goldman, Steven A.; Enikolopov, Grigori

2003-08-01

Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts

PubMed Central

Gurtan, Allan M.; Ravi, Arvind; Rahl, Peter B.; Bosson, Andrew D.; JnBaptiste, Courtney K.; Bhutkar, Arjun; Whittaker, Charles A.; Young, Richard A.; Sharp, Phillip A.

2013-01-01

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function. PMID:23630078

Association of Cell Adhesion Molecules Contactin-6 and Latrophilin-1 Regulates Neuronal Apoptosis

PubMed Central

Zuko, Amila; Oguro-Ando, Asami; Post, Harm; Taggenbrock, Renske L. R. E.; van Dijk, Roland E.; Altelaar, A. F. Maarten; Heck, Albert J. R.; Petrenko, Alexander G.; van der Zwaag, Bert; Shimoda, Yasushi; Pasterkamp, R. Jeroen; Burbach, J. Peter H.

2016-01-01

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment. PMID:28018171

Ballooning modes localized near the null point of a divertor

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

Farmer, W. A.; Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, California 94550

2014-04-15

The stability of ballooning modes localized to the null point in both the standard and snowflake divertors is considered. Ideal magnetohydrodynamics is used. A series expansion of the flux function is performed in the vicinity of the null point with the lowest, non-vanishing term retained for each divertor configuration. The energy principle is used with a trial function to determine a sufficient instability threshold. It is shown that this threshold depends on the orientation of the flux surfaces with respect to the major radius with a critical angle appearing due to the convergence of the field lines away from themore » null point. When the angle the major radius forms with respect to the flux surfaces exceeds this critical angle, the system is stabilized. Further, the scaling of the instability threshold with the aspect ratio and the ratio of the scrape-off-layer width to the major radius is shown. It is concluded that ballooning modes are not a likely candidate for driving convection in the vicinity of the null for parameters relevant to existing machines. However, the results place a lower bound on the width of the heat flux in the private flux region. To explain convective mixing in the vicinity of the null point, new consideration should be given to an axisymmetric mixing mode [W. A. Farmer and D. D. Ryutov, Phys. Plasmas 20, 092117 (2013)] as a possible candidate to explain current experimental results.« less

EFFECTS OF FIELD-LINE TOPOLOGY ON ENERGY PROPAGATION IN THE CORONA

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

Candelaresi, S.; Pontin, D. I.; Hornig, G.

We study the effect of photospheric footpoint motions on magnetic field structures containing magnetic nulls. The footpoint motions are prescribed on the photospheric boundary as a velocity field that entangles the magnetic field. We investigate the propagation of the injected energy, the conversion of energy, emergence of current layers, and other consequences of the nontrivial magnetic field topology in this situation. These boundary motions lead initially to an increase in magnetic and kinetic energy. Following this, the energy input from the photosphere is partially dissipated and partially transported out of the domain through the Poynting flux. The presence of separatrixmore » layers and magnetic null points fundamentally alters the propagation behavior of disturbances from the photosphere into the corona. Depending on the field-line topology close to the photosphere, the energy is either trapped or free to propagate into the corona.« less

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

Autism phenotypes in ZnT3 null mice: Involvement of zinc dyshomeostasis, MMP-9 activation and BDNF upregulation

PubMed Central

Yoo, Min Heui; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

2016-01-01

To investigate the role of synaptic zinc in the ASD pathogenesis, we examined zinc transporter 3 (ZnT3) null mice. At 4–5 weeks of age, male but not female ZnT3 null mice exhibited autistic-like behaviors. Cortical volume and neurite density were significantly greater in male ZnT3 null mice than in WT mice. In male ZnT3 null mice, consistent with enhanced neurotrophic stimuli, the level of BDNF as well as activity of MMP-9 was increased. Consistent with known roles for MMPs in BDNF upregulation, 2.5-week treatment with minocycline, an MMP inhibitor, significantly attenuated BDNF levels as well as megalencephaly and autistic-like behaviors. Although the ZnT3 null state removed synaptic zinc, it rather increased free zinc in the cytosol of brain cells, which appeared to increase MMP-9 activity and BDNF levels. The present results suggest that zinc dyshomeostasis during the critical period of brain development may be a possible contributing mechanism for ASD. PMID:27352957

Autism phenotypes in ZnT3 null mice: Involvement of zinc dyshomeostasis, MMP-9 activation and BDNF upregulation.

PubMed

Yoo, Min Heui; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

2016-06-29

To investigate the role of synaptic zinc in the ASD pathogenesis, we examined zinc transporter 3 (ZnT3) null mice. At 4-5 weeks of age, male but not female ZnT3 null mice exhibited autistic-like behaviors. Cortical volume and neurite density were significantly greater in male ZnT3 null mice than in WT mice. In male ZnT3 null mice, consistent with enhanced neurotrophic stimuli, the level of BDNF as well as activity of MMP-9 was increased. Consistent with known roles for MMPs in BDNF upregulation, 2.5-week treatment with minocycline, an MMP inhibitor, significantly attenuated BDNF levels as well as megalencephaly and autistic-like behaviors. Although the ZnT3 null state removed synaptic zinc, it rather increased free zinc in the cytosol of brain cells, which appeared to increase MMP-9 activity and BDNF levels. The present results suggest that zinc dyshomeostasis during the critical period of brain development may be a possible contributing mechanism for ASD.

Mesenchymal Stromal Cells Expressing Heme Oxygenase-1 Reverse Pulmonary Hypertension

PubMed Central

Liang, Olin D.; Mitsialis, S. Alex; Chang, Mun Seog; Vergadi, Eleni; Lee, Changjin; Aslam, Muhammad; Fernandez-Gonzalez, Angeles; Liu, Xianlan; Baveja, Rajiv; Kourembanas, Stella

2012-01-01

Pulmonary arterial hypertension (PAH) remains a serious disease, and, while current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically-modified mouse lines, we tested the ability of bone marrow-derived stromal cells (MSCs), to treat chronic hypoxia-induced PAH. Recipient mice were exposed for five weeks to normobaric hypoxia (8%–10% O2), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or Heme Oxygenase-1 (HO-1) null mice (Hmox1KO) conferred partial protection from PAH when transplanted into WT or Hmox1KO recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SHO1 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1KO animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only upon doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulative, our results demonstrate that MSCs ameliorate chronic hypoxia – induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1 dependent and HO-1 independent protective pathways. PMID:20957739

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

Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild typemore » of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells. • Overexpression of the Del-1 gene potentiates proliferation and invasion of lung carcinoma cells. • Del-1 may be used as a diagnostic or prognostic marker for lung cancer progression.« less

The barley anion channel, HvALMT1, has multiple roles in guard cell physiology and grain metabolism.

PubMed

Xu, Muyun; Gruber, Benjamin D; Delhaize, Emmanuel; White, Rosemary G; James, Richard A; You, Jiangfeng; Yang, Zhenming; Ryan, Peter R

2015-01-01

The barley (Hordeum vulgare) gene HvALMT1 encodes an anion channel in guard cells and in certain root tissues indicating that it may perform multiple roles. The protein localizes to the plasma membrane and facilitates malate efflux from cells when constitutively expressed in barley plants and Xenopus oocytes. This study investigated the function of HvALMT1 further by identifying its tissue-specific expression and by generating and characterizing RNAi lines with reduced HvALMT1 expression. We show that transgenic plants with 18-30% of wild-type HvALMT1 expression had impaired guard cell function. They maintained higher stomatal conductance in low light intensity and lost water more rapidly from excised leaves than the null segregant control plants. Tissue-specific expression of HvALMT1 was investigated in developing grain and during germination using transgenic barley lines expressing the green fluorescent protein (GFP) with the HvALMT1 promoter. We found that HvALMT1 is expressed in the nucellar projection, the aleurone layer and the scutellum of developing barley grain. Malate release measured from isolated aleurone layers prepared from imbibed grain was significantly lower in the RNAi barley plants compared with control plants. These data provide molecular and physiological evidence that HvALMT1 functions in guard cells, in grain development and during germination. We propose that HvALMT1 releases malate and perhaps other anions from guard cells to promote stomatal closure. The likely roles of HvALMT1 during seed development and grain germination are also discussed. © 2014 Scandinavian Plant Physiology Society.

Differential Radiosensitivity Phenotypes of DNA-PKcs Mutations Affecting NHEJ and HRR Systems following Irradiation with Gamma-Rays or Very Low Fluences of Alpha Particles

PubMed Central

Little, John B.; Kato, Takamitsu A.; Shih, Hung-Ying; Xie, Xian-Jin; Wilson Jr., Paul F.; Brogan, John R.; Kurimasa, Akihiro; Chen, David J.; Bedford, Joel S.; Chen, Benjamin P. C.

2014-01-01

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component. PMID:24714417

Impaired olfaction in mice lacking aquaporin-4 water channels

PubMed Central

Lu, Daniel C.; Zhang, Hua; Zador, Zsolt; Verkman, A. S.

2008-01-01

Aquaporin-4 (AQP4) is a water-selective transport protein expressed in glial cells throughout the central nervous system. AQP4 deletion in mice produces alterations in several neuroexcitation phenomena, including hearing, vision, epilepsy, and cortical spreading depression. Here, we report defective olfaction and electroolfactogram responses in AQP4-null mice. Immunofluorescence indicated strong AQP4 expression in supportive cells of the nasal olfactory epithelium. The olfactory epithelium in AQP4-null mice had identical appearance, but did not express AQP4, and had ∼12-fold reduced osmotic water permeability. Behavioral analysis showed greatly impaired olfaction in AQP4-null mice, with latency times of 17 ± 0.7 vs. 55 ± 5 s in wild-type vs. AQP4-null mice in a buried food pellet test, which was confirmed using an olfactory maze test. Electroolfactogram voltage responses to multiple odorants were reduced in AQP4-null mice, with maximal responses to triethylamine of 0.80 ± 0.07 vs. 0.28 ± 0.03 mV. Similar olfaction and electroolfactogram defects were found in outbred (CD1) and inbred (C57/bl6) mouse genetic backgrounds. Our results establish AQP4 as a novel determinant of olfaction, the deficiency of which probably impairs extracellular space K+ buffering in the olfactory epithelium.—Lu, D. C., Zhang, H., Zador, Z., Verkman, A. S. Impaired olfaction in mice lacking aquaporin-4 water channels. PMID:18511552

2010 August 1–2 Sympathetic Eruptions. II. Magnetic Topology of the MHD Background Field

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

Titov, Viacheslav S.; Mikić, Zoran; Török, Tibor

Using a potential field source-surface (PFSS) model, we recently analyzed the global topology of the background coronal magnetic field for a sequence of coronal mass ejections (CMEs) that occurred on 2010 August 1–2. Here we repeat this analysis for the background field reproduced by a magnetohydrodynamic (MHD) model that incorporates plasma thermodynamics. As for the PFSS model, we find that all three CME source regions contain a coronal hole (CH) that is separated from neighboring CHs by topologically very similar pseudo-streamer structures. However, the two models yield very different results for the size, shape, and flux of the CHs. Wemore » find that the helmet-streamer cusp line, which corresponds to a source-surface null line in the PFSS model, is structurally unstable and does not form in the MHD model. Our analysis indicates that, generally, in MHD configurations, this line instead consists of a multiple-null separator passing along the edge of disconnected-flux regions. Some of these regions are transient and may be the origin of the so-called streamer blobs. We show that the core topological structure of such blobs is a three-dimensional “plasmoid” consisting of two conjoined flux ropes of opposite handedness, which connect at a spiral null point of the magnetic field. Our analysis reveals that such plasmoids also appear in pseudo-streamers on much smaller scales. These new insights into the coronal magnetic topology provide some intriguing implications for solar energetic particle events and for the properties of the slow solar wind.« less

2010 August 1-2 Sympathetic Eruptions. II. Magnetic Topology of the MHD Background Field

NASA Astrophysics Data System (ADS)

Titov, Viacheslav S.; Mikić, Zoran; Török, Tibor; Linker, Jon A.; Panasenco, Olga

2017-08-01

Using a potential field source-surface (PFSS) model, we recently analyzed the global topology of the background coronal magnetic field for a sequence of coronal mass ejections (CMEs) that occurred on 2010 August 1-2. Here we repeat this analysis for the background field reproduced by a magnetohydrodynamic (MHD) model that incorporates plasma thermodynamics. As for the PFSS model, we find that all three CME source regions contain a coronal hole (CH) that is separated from neighboring CHs by topologically very similar pseudo-streamer structures. However, the two models yield very different results for the size, shape, and flux of the CHs. We find that the helmet-streamer cusp line, which corresponds to a source-surface null line in the PFSS model, is structurally unstable and does not form in the MHD model. Our analysis indicates that, generally, in MHD configurations, this line instead consists of a multiple-null separator passing along the edge of disconnected-flux regions. Some of these regions are transient and may be the origin of the so-called streamer blobs. We show that the core topological structure of such blobs is a three-dimensional “plasmoid” consisting of two conjoined flux ropes of opposite handedness, which connect at a spiral null point of the magnetic field. Our analysis reveals that such plasmoids also appear in pseudo-streamers on much smaller scales. These new insights into the coronal magnetic topology provide some intriguing implications for solar energetic particle events and for the properties of the slow solar wind.

[Recombinant adeno-associated virus mediated RNA interference of angiogenin expression inhibits cell growth of human lung adenocarcinoma].

PubMed

Li, Bai-Ling; Zhang, Guan-Xin; Hou, Xiao-Lei; Tan, Meng-Wei; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Huang, Sheng-Dong

2009-03-01

To study the inhibition of angiogenin (ANG) expression in human lung squamous cancer cell strain-A549 through adeno-associated virus (AAV)-mediated RNA-interference, and therefore to observe its effect on the growth of cancer cells and tumor formation. Recombinant AAV expressing H1-promoter-induced small-interference- RNA (siRNA) targeting ANG (AAV-shANG) was constructed, and then transfected into A549 cells. A549 cells and cells transfected with AAV-Null were used as the control groups. The effects of the reduced expression of ANG by RNAi from AAV-shANG on the growth, formation, reproduction, apoptosis, and microvessel-density of the carcinoma were observed. In vitro experiment showed that AAV-shANG was constructed successfully, There was an significant decrease in the expression of ANG protein 72 h after transfection, compared with the normal A459 cells and AAV-Null cells (P < 0.01). Cell cycle analysis showed that the proliferation index (PI) of normal A549 cells, AAV-Null cells and AAVshANG cells were 0.32 +/- 0.29, 0.35 +/- 0.38 and 0.31 +/- 0.43, respectively. There was no statistic difference in the PIs among the 3 groups (P > 0.05). In vivo experiment using thymus-defect mice showed that, there was an remarkable reduction in the mass and volume of tumors in AAV-shANG transfected group, compared to the control groups. Microvessel-density was 9.4 +/- 1.5, 9.8 +/- 2.1 and 5.7 +/- 1.9, respectively in the 3 groups, a statistic difference among the AAV-shANG-transfected group, the normal A549 group and the AAV-Null transfected group. The percentages of apoptotic cells in each group were (7.7 +/- 3.1)%, (8.5 +/- 5.4)%, (17.1 +/- 8.6)%, respectively, the experimental group being higher than those of the control groups. Positive rates of PCNA were (84.8 +/- 9.7)%, (85.8 +/- 9.8)%, and (70.4 +/- 10.1)%, respectively, the AAV-shANG transfected cancer cells showing a lower PCNA index than the control groups. AAV-mediated expression of siRNA could reduce the expression of ANG in cancer cells, significantly enough to inhibit cell proliferation, promote cell apoptosis and inhibit tumor growth.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts

PubMed Central

Hagos, Engda G; Ghaleb, Amr M; Kumar, Amrita; Neish, Andrew S; Yang, Vincent W

2011-01-01

Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions. PMID:21892412

Neto Auxiliary Protein Interactions Regulate Kainate and NMDA Receptor Subunit Localization at Mossy Fiber–CA3 Pyramidal Cell Synapses

PubMed Central

Wyeth, Megan S.; Pelkey, Kenneth A.; Petralia, Ronald S.; Salter, Michael W.; McInnes, Roderick R.

2014-01-01

Neto1 and Neto2 auxiliary subunits coassemble with NMDA receptors (NMDARs) and kainate receptors (KARs) to modulate their function. In the hippocampus, Neto1 enhances the amplitude and prolongs the kinetics of KAR-mediated currents at mossy fiber (MF)–CA3 pyramidal cell synapses. However, whether Neto1 trafficks KARs to synapses or simply alters channel properties is unresolved. Therefore, postembedding electron microscopy was performed to investigate the localization of GluK2/3 subunits at MF–CA3 synapses in Neto-null mice. Postsynaptic GluK2/3 Immunogold labeling was substantially reduced in Neto-null mice compared with wild types. Moreover, spontaneous KAR-mediated synaptic currents and metabotropic KAR signaling were absent in CA3 pyramidal cells of Neto-null mice. A similar loss of ionotropic and metabotropic KAR function was observed in Neto1, but not Neto2, single knock-out mice, specifically implicating Neto1 in regulating CA3 pyramidal cell KAR localization and function. Additional controversy pertains to the role of Neto proteins in modulating synaptic NMDARs. While Immunogold labeling for GluN2A at MF–CA3 synapses was comparable between wild-type and Neto-null mice, labeling for postsynaptic GluN2B was robustly increased in Neto-null mice. Accordingly, NMDAR-mediated currents at MF–CA3 synapses exhibited increased sensitivity to a GluN2B-selective antagonist in Neto1 knockouts relative to wild types. Thus, despite preservation of the overall MF–CA3 synaptic NMDAR-mediated current, loss of Neto1 alters NMDAR subunit composition. These results confirm that Neto protein interactions regulate synaptic localization of KAR and NMDAR subunits at MF–CA3 synapses, with implications for both ionotropic and metabotropic glutamatergic recruitment of the CA3 network. PMID:24403160

Selective depletion of microglial progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation.

PubMed

Petkau, Terri L; Kosior, Natalia; de Asis, Kathleen; Connolly, Colúm; Leavitt, Blair R

2017-11-17

Progranulin deficiency due to heterozygous null mutations in the GRN gene are a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations are thought to be a rare cause of neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout (Grn-null) mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. In the brain, progranulin is predominantly expressed in neurons and microglia, and previously, we demonstrated that neuronal-specific depletion of progranulin does not recapitulate the neuropathological phenotype of Grn-null mice. In this study, we evaluated whether selective depletion of progranulin expression in myeloid-lineage cells, including microglia, causes NCL-like neuropathology or neuroinflammation in mice. We generated mice with progranulin depleted in myeloid-lineage cells by crossing mice homozygous for a floxed progranulin allele to mice expressing Cre recombinase under control of the LyzM promotor (Lyz-cKO). Progranulin expression was reduced by approximately 50-70% in isolated microglia compared to WT levels. Lyz-cKO mice aged to 12 months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for many of these measures in Grn-null animals. To evaluate the functional effect of reduced progranulin expression in isolated microglia, primary cultures were stimulated with controlled standard endotoxin and cytokine release was measured. While Grn-null microglia display a hyper-inflammatory phenotype, Lyz-cKO and WT microglia secreted similar levels of inflammatory cytokines. We conclude that progranulin expression from either microglia or neurons is sufficient to prevent the development of NCL-like neuropathology in mice. Furthermore, microglia that are deficient for progranulin expression but isolated from a progranulin-rich environment have a normal inflammatory profile. Our results suggest that progranulin acts, at least partly, in a non-cell autonomous manner in the brain.

SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

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

Vachtenheim, Jiri, E-mail: jivach@upn.anet.cz; Ondrusova, Lubica; Borovansky, Jan

2010-02-12

The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brmmore » was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.« less

p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer.

PubMed

Tonnessen-Murray, Crystal; Ungerleider, Nathan A; Rao, Sonia G; Wasylishen, Amanda R; Frey, Wesley D; Jackson, James G

2018-05-28

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum

PubMed Central

Platt, James L.; Rogers, Benjamin J.; Rogers, Kelley C.; Harwood, Adrian J.; Kimmel, Alan R.

2013-01-01

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression. PMID:24301467

PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines

PubMed Central

Ginés, Alba; Bystrup, Sara; Ruiz de Porras, Vicenç; Guardia, Cristina; Musulén, Eva; Martínez-Cardús, Anna; Manzano, José Luis; Layos, Laura; Abad, Albert; Martínez-Balibrea, Eva

2015-01-01

Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells. PMID:25955657

Nulling at the Keck Interferometer

NASA Technical Reports Server (NTRS)

Colavita, M. Mark; Serabyn, Gene; Wizinowich, Peter L.; Akeson, Rachel L.

2006-01-01

The nulling mode of the Keck Interferometer is being commissioned at the Mauna Kea summit. The nuller combines the two Keck telescope apertures in a split-pupil mode to both cancel the on-axis starlight and to coherently detect the residual signal. The nuller, working at 10 um, is tightly integrated with the other interferometer subsystems including the fringe and angle trackers, the delay lines and laser metrology, and the real-time control system. Since first 10 um light in August 2004, the system integration is proceeding with increasing functionality and performance, leading to demonstration of a 100:1 on-sky null in 2005. That level of performance has now been extended to observations with longer coherent integration times. An overview of the overall system is presented, with emphasis on the observing sequence, phasing system, and differences with respect to the V2 system, along with a presentation of some recent engineering data.

High-frequency waves in the corona due to null points

NASA Astrophysics Data System (ADS)

Santamaria, I. C.; Khomenko, E.; Collados, M.; de Vicente, A.

2017-06-01

This work aims to understand the behavior of non-linear waves in the vicinity of a coronal null point. In previous works we have shown that high-frequency waves are generated in such a magnetic configuration. This paper studies those waves in detail in order to provide a plausible explanation of their generation. We demonstrate that slow magneto-acoustic shock waves generated in the chromosphere propagate through the null point and produce a train of secondary shocks that escape along the field lines. A particular combination of the shock wave speeds generates waves at a frequency of 80 mHz. We speculate that this frequency may be sensitive to the atmospheric parameters in the corona and therefore can be used to probe the structure of this solar layer. Movies attached to Figs 2 and 4 are available at http://www.aanda.org

CXC Chemokine Receptor 7 (CXCR7) Regulates CXCR4 Protein Expression and Capillary Tuft Development in Mouse Kidney

PubMed Central

Haege, Sammy; Mueller, Wiebke; Nietzsche, Sandor; Lupp, Amelie; Mackay, Fabienne; Schulz, Stefan; Stumm, Ralf

2012-01-01

Background The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development. Methodology/Principal Findings We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping. Conclusions/Significance We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries. PMID:22880115

CXC chemokine receptor 7 (CXCR7) regulates CXCR4 protein expression and capillary tuft development in mouse kidney.

PubMed

Haege, Sammy; Einer, Claudia; Thiele, Stefanie; Mueller, Wiebke; Nietzsche, Sandor; Lupp, Amelie; Mackay, Fabienne; Schulz, Stefan; Stumm, Ralf

2012-01-01

The CXCL12/CXCR4 axis is involved in kidney development by regulating formation of the glomerular tuft. Recently, a second CXCL12 receptor was identified and designated CXCR7. Although it is established that CXCR7 regulates heart and brain development in conjunction with CXCL12 and CXCR4, little is known about the influence of CXCR7 on CXCL12 dependent kidney development. We provided analysis of CXCR7 expression and function in the developing mouse kidney. Using in situ hybridization, we identified CXCR7 mRNA in epithelial cells including podocytes at all nephron stages up to the mature glomerulus. CXCL12 mRNA showed a striking overlap with CXCR7 mRNA in epithelial structures. In addition, CXCL12 was detected in stromal cells and the glomerular tuft. Expression of CXCR4 was complementary to that of CXCR7 as it occurred in mesenchymal cells, outgrowing ureteric buds and glomerular endothelial cells but not in podocytes. Kidney examination in CXCR7 null mice revealed ballooning of glomerular capillaries as described earlier for CXCR4 null mice. Moreover, we detected a severe reduction of CXCR4 protein but not CXCR4 mRNA within the glomerular tuft and in the condensed mesenchyme. Malformation of the glomerular tuft in CXCR7 null mice was associated with mesangial cell clumping. We established that there is a similar glomerular pathology in CXCR7 and CXCR4 null embryos. Based on the phenotype and the anatomical organization of the CXCL12/CXCR4/CXCR7 system in the forming glomerulus, we propose that CXCR7 fine-tunes CXCL12/CXCR4 mediated signalling between podocytes and glomerular capillaries.

Mechanism of Growth Inhibition of Human Cancer Cells by Conjugated Eicosapentaenoic Acid, an Inhibitor of DNA Polymerase and Topoisomerase

PubMed Central

Yonezawa, Yuko; Yoshida, Hiromi; Mizushina, Yoshiyuki

2007-01-01

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We found that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) inhibited the activities of eukaryotic pols and topos in vitro, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC50 values for mammalian pols and human topos of 11.0 – 31.8 and 0.5 – 2.5 μM, respectively. cEPA inhibited the proliferation of two human leukemia cell lines, NALM-6, which is a p53-wild type, and HL-60, which is a p53-null mutant, and the inhibitory effect was stronger than that of normal EPA. In both cell lines, cEPA arrested in the G1 phase, and increased cyclin E protein levels, indicating that it blocks the primary step of in vivo DNA replication by inhibiting the activity of replicative pols rather than topos. DNA replication-related proteins, such as RPA70, ATR and phosphorylated-Chk1/2, were increased by cEPA treatment in the cell lines, suggesting that cEPA led to DNA replication fork stress inhibiting the activities of pols and topos, and the ATR-dependent DNA damage response pathway could respond to the inhibitor of DNA replication. The compound induced cell apoptosis through both p53-dependent and p53-independent pathways in cell lines NALM-6 and HL-60, respectively. These results suggested the therapeutic potential of conjugated PUFA, such as cEPA, as a leading anti-cancer compound that inhibited pols and topos activities.

Genetic disruption of SOD1 gene causes glucose intolerance and impairs β-cell function.

PubMed

Muscogiuri, Giovanna; Salmon, Adam B; Aguayo-Mazzucato, Cristina; Li, Mengyao; Balas, Bogdan; Guardado-Mendoza, Rodolfo; Giaccari, Andrea; Reddick, Robert L; Reyna, Sara M; Weir, Gordon; Defronzo, Ralph A; Van Remmen, Holly; Musi, Nicolas

2013-12-01

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased β-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow-fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction.

Genetic Disruption of SOD1 Gene Causes Glucose Intolerance and Impairs β-Cell Function

PubMed Central

Muscogiuri, Giovanna; Salmon, Adam B.; Aguayo-Mazzucato, Cristina; Li, Mengyao; Balas, Bogdan; Guardado-Mendoza, Rodolfo; Giaccari, Andrea; Reddick, Robert L.; Reyna, Sara M.; Weir, Gordon; DeFronzo, Ralph A.; Van Remmen, Holly; Musi, Nicolas

2013-01-01

Oxidative stress has been associated with insulin resistance and type 2 diabetes. However, it is not clear whether oxidative damage is a cause or a consequence of the metabolic abnormalities present in diabetic subjects. The goal of this study was to determine whether inducing oxidative damage through genetic ablation of superoxide dismutase 1 (SOD1) leads to abnormalities in glucose homeostasis. We studied SOD1-null mice and wild-type (WT) littermates. Glucose tolerance was evaluated with intraperitoneal glucose tolerance tests. Peripheral and hepatic insulin sensitivity was quantitated with the euglycemic-hyperinsulinemic clamp. β-Cell function was determined with the hyperglycemic clamp and morphometric analysis of pancreatic islets. Genetic ablation of SOD1 caused glucose intolerance, which was associated with reduced in vivo β-cell insulin secretion and decreased β-cell volume. Peripheral and hepatic insulin sensitivity were not significantly altered in SOD1-null mice. High-fat diet caused glucose intolerance in WT mice but did not further worsen the glucose intolerance observed in standard chow–fed SOD1-null mice. Our findings suggest that oxidative stress per se does not play a major role in the pathogenesis of insulin resistance and demonstrate that oxidative stress caused by SOD1 ablation leads to glucose intolerance secondary to β-cell dysfunction. PMID:24009256

RhoE deficiency alters postnatal subventricular zone development and the number of calbindin-expressing neurons in the olfactory bulb of mouse.

PubMed

Ballester-Lurbe, Begoña; González-Granero, Susana; Mocholí, Enric; Poch, Enric; García-Manzanares, María; Dierssen, Mara; Pérez-Roger, Ignacio; García-Verdugo, José M; Guasch, Rosa M; Terrado, José

2015-11-01

The subventricular zone represents an important reservoir of progenitor cells in the adult brain. Cells from the subventricular zone migrate along the rostral migratory stream and reach the olfactory bulb, where they originate different types of interneurons. In this work, we have analyzed the role of the small GTPase RhoE/Rnd3 in subventricular zone cell development using mice-lacking RhoE expression. Our results show that RhoE null mice display a remarkable postnatal broadening of the subventricular zone and caudal rostral migratory stream. This broadening was caused by an increase in progenitor proliferation, observed in the second postnatal week but not before, and by an altered migration of the cells, which appeared in disorganized cell arrangements that impaired the appropriate contact between cells in the rostral migratory stream. In addition, the thickness of the granule cell layer in the olfactory bulb was reduced, although the density of granule cells did not differ between wild-type and RhoE null mice. Finally, the lack of RhoE expression affected the olfactory glomeruli inducing a severe reduction of calbindin-expressing interneurons in the periglomerular layer. This was already evident in the newborns and even more pronounced 15 days later when RhoE null mice displayed 89% less cells than control mice. Our results indicate that RhoE has pleiotropic functions on subventricular cells because of its role in proliferation and tangential migration, affecting mainly the development of calbindin-expressing cells in the olfactory bulb.

Altered Expression of the Malate-Permeable Anion Channel OsALMT4 Reduces the Growth of Rice Under Low Radiance

PubMed Central

Liu, Jie; Xu, Muyun; Estavillo, Gonzalo M.; Delhaize, Emmanuel; White, Rosemary G.; Zhou, Meixue; Ryan, Peter R.

2018-01-01

We examined the function of OsALMT4 in rice (Oryza sativa L.) which is a member of the aluminum-activated malate transporter family. Previous studies showed that OsALMT4 localizes to the plasma membrane and that expression in transgenic rice lines results in a constitutive release of malate from the roots. Here, we show that OsALMT4 is expressed widely in roots, shoots, flowers, and grain but not guard cells. Expression was also affected by ionic and osmotic stress, light and to the hormones ABA, IAA, and salicylic acid. Malate efflux from the transgenic plants over-expressing OsALMT4 was inhibited by niflumate and salicylic acid. Growth of transgenic lines with either increased OsALMT4 expression or reduced expression was measured in different environments. Light intensity caused significant differences in growth between the transgenic lines and controls. When day-time light was reduced from 700 to 300 μmol m-2s-1 independent transgenic lines with either increased or decreased OsALMT4 expression accumulated less biomass compared to their null controls. This response was not associated with differences in photosynthetic capacity, stomatal conductance or sugar concentrations in tissues. We propose that by disrupting malate fluxes across the plasma membrane carbon partitioning and perhaps signaling are affected which compromises growth under low light. We conclude that OsALMT4 is expressed widely in rice and facilitates malate efflux from different cell types. Altering OsALMT4 expression compromises growth in low-light environments. PMID:29774038

Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage.

PubMed

Cui, Hongmei; Gu, Xinsheng; Chen, Jingshu; Xie, Ying; Ke, Sui; Wu, Jing; Golovko, Andrei; Morpurgo, Benjamin; Yan, Chunhong; Phillips, Timothy D; Xie, Wen; Luo, Jianyuan; Zhou, Zhijun; Tian, Yanan

2017-06-05

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation. Copyright © 2017 Elsevier B.V. All rights reserved.

Ginsenoside metabolite compound K enhances the efficacy of cisplatin in lung cancer cells.

PubMed

Li, Yang; Zhou, Tong; Ma, Chengyuan; Song, Weiwei; Zhang, Jian; Yu, Zhenxiang

2015-03-01

To evaluate the potential of ginsenoside metabolite compound K (CK) in enhancing the anti-tumor effects of cisplatin against lung cancer cells, including cell proliferation and apoptosis, and the underlying mechanism. Western blotting and p53 reporter assay were used to assess p53 expression and activity. MTT assay and TUNEL staining were employed to investigate the drug effects on cell growth and apoptosis, respectively. Combination index (CI) was calculated to determine synergism. We found that CK could significantly enhance cisplatin-induced p53 expression and activity in two lung cancer cell lines, H460 and A549. Consequently, synergistic inhibition of cell growth was observed when the cells were co-treated with CK and cisplatin compared to single treatment. In addition, the ability of cisplatin in apoptosis induction was similarly synergized by CK. Furthermore, by using p53-null lung cancer cells, we demonstrate that the synergy was p53 dependent. Conventional chemotherapies are often accompanied by development of drug resistance and severe side effects. Novel discoveries of low toxicity compounds to improve the outcome or enhance the efficacy of chemotherapies are of great interest. In the present study, our data provide the first evidence that CK could be potentially used as an agent to synergize the efficacy of cisplatin in lung cancer.

Telomere lengthening early in development.

PubMed

Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

2007-12-01

Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

The C. elegans VAPB homolog VPR-1 is a permissive signal for gonad development.

PubMed

Cottee, Pauline A; Cole, Tim; Schultz, Jessica; Hoang, Hieu D; Vibbert, Jack; Han, Sung Min; Miller, Michael A

2017-06-15

VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis. © 2017. Published by The Company of Biologists Ltd.

The C. elegans VAPB homolog VPR-1 is a permissive signal for gonad development

PubMed Central

Cole, Tim; Hoang, Hieu D.; Han, Sung Min

2017-01-01

VAMP/synaptobrevin-associated proteins (VAPs) contain an N-terminal major sperm protein domain (MSPd) that is associated with amyotrophic lateral sclerosis. VAPs have an intracellular housekeeping function, as well as an extracellular signaling function mediated by the secreted MSPd. Here we show that the C. elegans VAP homolog VPR-1 is essential for gonad development. vpr-1 null mutants are maternal effect sterile due to arrested gonadogenesis following embryo hatching. Somatic gonadal precursor cells and germ cells fail to proliferate fully and complete their respective differentiation programs. Maternal or zygotic vpr-1 expression is sufficient to induce gonadogenesis and fertility. Genetic mosaic and cell type-specific expression studies indicate that vpr-1 activity is important in the nervous system, germ line and intestine. VPR-1 acts in parallel to Notch signaling, a key regulator of germline stem cell proliferation and differentiation. Neuronal vpr-1 expression is sufficient for gonadogenesis induction during a limited time period shortly after hatching. These results support the model that the secreted VPR-1 MSPd acts at least in part on gonadal sheath cell precursors in L1 to early L2 stage hermaphrodites to permit gonadogenesis. PMID:28634273

Development of versatile non-homologous end joining-based knock-in module for genome editing.

PubMed

Sawatsubashi, Shun; Joko, Yudai; Fukumoto, Seiji; Matsumoto, Toshio; Sugano, Shigeo S

2018-01-12

CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.

Therapeutic Role of Rifaximin in Inflammatory Bowel Disease: Clinical Implication of Human Pregnane X Receptor Activation

PubMed Central

Cheng, Jie; Shah, Yatrik M.; Ma, Xiaochao; Pang, Xiaoyan; Tanaka, Toshiya; Kodama, Tatsuhiko; Krausz, Kristopher W.

2010-01-01

Human pregnane X receptor (PXR) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a human PXR activator, is in clinical trials for treatment of IBD and has demonstrated efficacy in Crohn's disease and active ulcerative colitis. In the current study, the protective and therapeutic role of rifaximin in IBD and its respective mechanism were investigated. PXR-humanized (hPXR), wild-type, and Pxr-null mice were treated with rifaximin in the dextran sulfate sodium (DSS)-induced and trinitrobenzene sulfonic acid (TNBS)-induced IBD models to determine the protective function of human PXR activation in IBD. The therapeutic role of rifaximin was further evaluated in DSS-treated hPXR and Pxr-null mice. Results demonstrated that preadministration of rifaximin ameliorated the clinical hallmarks of colitis in DSS- and TNBS-treated hPXR mice as determined by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. In addition, higher survival rates and recovery from colitis symptoms were observed in hPXR mice, but not in Pxr-null mice, when rifaximin was administered after the onset of symptoms. Nuclear factor κB (NF-κB) target genes were markedly down-regulated in hPXR mice by rifaximin treatment. In vitro NF-κB reporter assays demonstrated inhibition of NF-κB activity after rifaximin treatment in colon-derived cell lines expressing hPXR. These findings demonstrated the preventive and therapeutic role of rifaximin on IBD through human PXR-mediated inhibition of the NF-κB signaling cascade, thus suggesting that human PXR may be an effective target for the treatment of IBD. PMID:20627999

Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of a GATA1 Protein Lacking the N-terminal Domain*

PubMed Central

Kobayashi, Eri; Shimizu, Ritsuko; Kikuchi, Yuko; Takahashi, Satoru; Yamamoto, Masayuki

2010-01-01

GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene. PMID:19854837

Premature cell senescence and T cell receptor-independent activation of CD8T cells in Juvenile Idiopathic Arthritis*

PubMed Central

Dvergsten, Jeffrey A.; Mueller, Robert G.; Griffin, Patricia; Abedin, Sameem; Pishko, Allyson; Michel, Joshua J.; Rosenkranz, Margalit E.; Reed, Ann M.; Kietz, Daniel A.; Vallejo, Abbe N.

2013-01-01

Objectives CD8T cells lacking CD28 were originally reported by Wedderburn and colleagues as a characteristic feature of JIA, but the relevance of these unusual cells to JIA remains to be elucidated. Because of recent evidence that CD28 loss is typical of terminally differentiated lymphocytes, we examined for functional subsets of CD8T cells in JIA. Methods Following informed consent/assent, blood and/or waste synovial fluid were collected from children with definite diagnosis of JIA (n = 98). De-identified blood (n = 33) and cord blood (n = 13) samples from healthy donors were also collected. CD8T and CD4T cells were screened for novel receptors, and where indicated, bioassays were performed to determine functional relevance of the identified receptor. Results Patients had a naïve T cell compartment with shortened telomeres, and their entire T cell pool had reduced proliferative capacity. They had an over abundance of CD31+CD28null CD8T cells, which was a significant feature of oligoarticular JIA (n = 62) compared to polyarticular JIA (n = 36). CD31+CD28null CD8T cells had limited mitotic capacity, and expressed high levels of the senescence antigens γH2Ax and/or p16. Ligation of CD31, independent of the TCR, sufficiently induced tyrosine phosphorylation, vesicle exocytosis, and production of IFN-γ and IL-10. Conclusion These data provide the first evidence for cell senescence, represented by CD31+CD28null CD8T cells, in the pathophysiology of JIA. Activation of these unusual cells in a TCR-independent manner suggests they are maladaptive, and could be potential targets for immunotherapy. PMID:23686519

Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling

PubMed Central

Hoffman, Laura M.; Jensen, Christopher C.; Kloeker, Susanne; Wang, C.-L. Albert; Yoshigi, Masaaki; Beckerle, Mary C.

2006-01-01

Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, we characterized the phenotype of fibroblasts in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. We identified differences in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. We postulate that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling. PMID:16505170

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration.

PubMed

Martínez-Sarrà, Ester; Montori, Sheyla; Gil-Recio, Carlos; Núñez-Toldrà, Raquel; Costamagna, Domiziana; Rotini, Alessio; Atari, Maher; Luttun, Aernout; Sampaolesi, Maurilio

2017-07-27

Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206 + macrophages. Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.

E-Cadherin Acts as a Regulator of Transcripts Associated with a Wide Range of Cellular Processes in Mouse Embryonic Stem Cells

PubMed Central

Soncin, Francesca; Mohamet, Lisa; Ritson, Sarah; Hawkins, Kate; Bobola, Nicoletta; Zeef, Leo; Merry, Catherine L. R.; Ward, Christopher M.

2011-01-01

Background We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES) cells. Methodology In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/-) ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3. Results We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional β-catenin pool that is able to induce β-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous β-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of β-catenin/TCF transactivation. PMID:21779327

E-cadherin acts as a regulator of transcripts associated with a wide range of cellular processes in mouse embryonic stem cells.

PubMed

Soncin, Francesca; Mohamet, Lisa; Ritson, Sarah; Hawkins, Kate; Bobola, Nicoletta; Zeef, Leo; Merry, Catherine L R; Ward, Christopher M

2011-01-01

We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES) cells. In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/-) ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3. We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional β-catenin pool that is able to induce β-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous β-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of β-catenin/TCF transactivation.

Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak

NASA Astrophysics Data System (ADS)

Zheng, Jing-Yi; Boustany, Nada N.

2010-07-01

Optical scatter imaging is used to estimate organelle size distributions in immortalized baby mouse kidney cells treated with 0.4 μM staurosporine to induce apoptosis. The study comprises apoptosis competent iBMK cells (W2) expressing the proapoptotic proteins Bax/Bak, apoptosis resistant Bax/Bak null cells (D3), and W2 and D3 cells expressing yellow fluorescent protein (YFP) or YFP fused to the antiapoptotic protein Bcl-xL (YFP-Bcl-xL). YFP expression is diffuse within the transfected cells, while YFP-Bcl-xL is localized to the mitochondria. Our results show a significant increase in the mean subcellular particle size from approximately 1.1 to 1.4 μm in both Bax/Bak expressing and Bax/Bak null cells after 60 min of STS treatment compared to DMSO-treated control cells. This dynamic is blocked by overexpression of YFP-Bcl-xL in Bax/Bak expressing cells, but is less significantly inhibited by YFP-Bcl-xL in Bax/Bak null cells. Our data suggest that the increase in subcellular particle size at the onset of apoptosis is modulated by Bcl-xL in the presence of Bax/Bak, but it occurs upstream of the final commitment to programmed cell death. Mitochondrial localization of YFP-Bcl-xL and the finding that micron-sized particles give rise to the scattering signal further suggest that alterations in mitochondrial morphology may underlie the observed changes in light scattering.

Lack of association between Kidd blood group system and chronic kidney disease.

PubMed

Capriolli, Tiago Verri; Visentainer, Jeane Eliete Laguila; Sell, Ana Maria

The Kidd blood group system has three antigens, Jk a , Jk b and Jk3, found on red blood cells and on endothelial cells of the inner lining of blood vessels in the renal medulla. These are known as urea transporter B (UT-B). Researchers have found that individuals carrying the Jk(a-b-) or Jk-null (UT-B null) phenotypes have a lower urine-concentrating capability and risk of severe renal impairment. This study evaluated the distribution of the Kidd phenotypes in patients with chronic kidney disease and a possible association of Kidd antigens with the development of renal disease. Jk a and Jk b antigens were phenotyped using the gel column agglutination test (ID-cards Bio-RAD) in 197 patients with chronic kidney disease and 444 blood donors, as the control group. The phenotype and antigen frequencies between patients and controls were evaluated using the Chi-square method with Yates correction and logistic regression after adjustments for gender and age. No differences were observed between the Kidd phenotypes frequency distribution between patients with chronic kidney disease and blood donors [Jk(a-b+)=22.3% and 27.2%; Jk(a+b-)=30.5% and 24.3%; Jk(a+b+)=47.25% and 48.4%, respectively]. The distribution of Kidd phenotypes found in the studied population is expected for Caucasians; Jk a and Jk b antigens and phenotypes were not found to be related to susceptibility for chronic kidney disease. Copyright © 2017 Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. All rights reserved.

In-situ observations of flux ropes formed in association with a pair of spiral nulls in magnetotail plasmas

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

Guo, Ruilong; Xie, Lun; He, Jiansen

Signatures of secondary islands are frequently observed in the magnetic reconnection regions of magnetotail plasmas. In this paper, magnetic structures with the secondary-island signatures observed by Cluster are reassembled by a fitting-reconstruction method. The results show three-dimensionally that a secondary island event can manifest the flux rope formed with an A{sub s}-type null and a B{sub s}-type null paired via their spines. We call this A{sub s}-spine-B{sub s}-like configuration the helically wrapped spine model. The reconstructed field lines wrap around the spine to form the flux rope, and an O-type topology is therefore seen on the plane perpendicular to themore » spine. Magnetized electrons are found to rotate on and cross the fan surface, suggesting that both the torsional-spine and the spine-fan reconnection take place in the configuration. Furthermore, detailed analysis implies that the spiral nulls and flux ropes were locally generated nearby the spacecraft in the reconnection outflow region, indicating that secondary reconnection may occur in the exhaust away from the primary reconnection site.« less

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function

PubMed Central

Chawla, Amanpreet Singh; Agrawal, Tanvi; Biswas, Moanaro; Vrati, Sudhanshu; Rath, Satyajit; George, Anna; Medigeshi, Guruprasad R.

2017-01-01

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β–T cells (TCRβ–null) are highly susceptible and die over 10–18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage. PMID:28151989

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

PubMed

Jain, Nidhi; Oswal, Neelam; Chawla, Amanpreet Singh; Agrawal, Tanvi; Biswas, Moanaro; Vrati, Sudhanshu; Rath, Satyajit; George, Anna; Bal, Vineeta; Medigeshi, Guruprasad R

2017-02-01

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β-T cells (TCRβ-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

Identification of CD166 as a Surface Marker for Enriching Prostate Stem/Progenitor and Cancer Initiating Cells

PubMed Central

Wang, Shunyou; Tran, Linh M.; Goldstein, Andrew S.; Lawson, Devon; Chen, Donghui; Li, Yunfeng; Guo, Changyong; Zhang, Baohui; Fazli, Ladan; Gleave, Martin; Witte, Owen N.; Garraway, Isla P.; Wu, Hong

2012-01-01

New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin-;Sca1+;CD49fhi (LSChi), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSChi subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSChi and Pten null LSChi. Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics. PMID:22880034

Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion cells lack direction tuning.

PubMed

Park, Silvia J H; Kim, In-Jung; Looger, Loren L; Demb, Jonathan B; Borghuis, Bart G

2014-03-12

Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation

PubMed Central

Apostolidis, Pani A.; Lindsey, Stephan; Miller, William M.

2012-01-01

During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects. PMID:22548738

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

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

Chhipa, Rishi Raj; Kumari, Ratna; Upadhyay, Ankur Kumar

2007-11-15

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expressionmore » levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.« less

T cell PPARγ is required for the anti-inflammatory efficacy of abscisic acid against experimental IBD.

PubMed

Guri, Amir J; Evans, Nicholas P; Hontecillas, Raquel; Bassaganya-Riera, Josep

2011-09-01

The phytohormone abscisic acid (ABA) has been shown to be effective in ameliorating chronic and acute inflammation. The objective of this study was to investigate whether ABA's anti-inflammatory efficacy in the gut is dependent on peroxisome proliferator-activated receptor γ (PPARγ) in T cells. PPARγ-expressing and T cell-specific PPARγ null mice were fed diets with or without ABA (100 mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate. The severity of clinical disease was assessed daily, and mice were euthanized on Day 7 of the dextran sodium sulfate challenge. Colonic inflammation was assessed through macroscopic and histopathological examination of inflammatory lesions and real-time quantitative RT-PCR-based quantification of inflammatory genes. Flow cytometry was used to phenotypically characterize leukocyte populations in the blood and mesenteric lymph nodes. Colonic sections were stained immunohistochemically to determine the effect of ABA on colonic regulatory T (T(reg)) cells. ABA's beneficial effects on disease activity were completely abrogated in T cell-specific PPARγ null mice. Additionally, ABA improved colon histopathology, reduced blood F4/80(+)CD11b(+) monocytes, increased the percentage of CD4(+) T cells expressing the inhibitory molecule cytotoxic T lymphocyte antigen 4 in blood and enhanced the number of T(reg) cells in the mesenteric lymph nodes and colons of PPARγ-expressing but not T cell-specific PPARγ null mice. We conclude that dietary ABA ameliorates experimental inflammatory bowel disease by enhancing T(reg) cell accumulation in the colonic lamina propria through a PPARγ-dependent mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.

Left cardiac isomerism in the Sonic hedgehog null mouse.

PubMed

Hildreth, Victoria; Webb, Sandra; Chaudhry, Bill; Peat, Jonathan D; Phillips, Helen M; Brown, Nigel; Anderson, Robert H; Henderson, Deborah J

2009-06-01

Sonic hedgehog (Shh) is a secreted morphogen necessary for the production of sidedness in the developing embryo. In this study, we describe the morphology of the atrial chambers and atrioventricular junctions of the Shh null mouse heart. We demonstrate that the essential phenotypic feature is isomerism of the left atrial appendages, in combination with an atrioventricular septal defect and a common atrioventricular junction. These malformations are known to be frequent in humans with left isomerism. To confirm the presence of left isomerism, we show that Pitx2c, a recognized determinant of morphological leftness, is expressed in the Shh null mutants on both the right and left sides of the inflow region, and on both sides of the solitary arterial trunk exiting from the heart. It has been established that derivatives of the second heart field expressing Isl1 are asymmetrically distributed in the developing normal heart. We now show that this population is reduced in the hearts from the Shh null mutants, likely contributing to the defects. To distinguish the consequences of reduced contributions from the second heart field from those of left-right patterning disturbance, we disrupted the movement of second heart field cells into the heart by expressing dominant-negative Rho kinase in the population of cells expressing Isl1. This resulted in absence of the vestibular spine, and presence of atrioventricular septal defects closely resembling those seen in the hearts from the Shh null mutants. The primary atrial septum, however, was well formed, and there was no evidence of isomerism of the atrial appendages, suggesting that these features do not relate to disruption of the contributions made by the second heart field. We demonstrate, therefore, that the Shh null mouse is a model of isomerism of the left atrial appendages, and show that the recognized associated malformations found at the venous pole of the heart in the setting of left isomerism are likely to arise from the loss of the effects of Shh in the establishment of laterality, combined with a reduced contribution made by cells derived from the second heart field.

ABCC4 Is a Determinant of Cytarabine‐Induced Cytotoxicity and Myelosuppression

PubMed Central

Drenberg, CD; Hu, S; Li, L; Buelow, DR; Orwick, SJ; Gibson, AA; Schuetz, JD; Sparreboom, A

2016-01-01

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4‐mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4‐expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4‐null mice, cytarabine‐induced hematological toxicity was enhanced and ex vivo colony‐forming assays showed that Abcc4‐deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine‐mediated insults in leukemic and host myeloid cells. PMID:26842729

The Spectrum of WRN Mutations in Werner Syndrome Patients

PubMed Central

Huang, Shurong; Lee, Lin; Hanson, Nancy B.; Lenaerts, Catherine; Hoehn, Holger; Poot, Martin; Rubin, Craig D.; Chen, Da-Fu; Yang, Chih-Chao; Juch, Heike; Dorn, Thomas; Spiegel, Roland; Oral, Elif Arioglu; Abid, Mohammed; Battisti, Carla; Lucci-Cordisco, Emanuela; Neri, Giovanni; Steed, Erin H.; Kidd, Alexa; Isley, William; Showalter, David; Vittone, Janet L.; Konstantinow, Alexander; Ring, Johannes; Meyer, Peter; Wenger, Sharon L.; von Herbay, Axel; Wollina, Uwe; Schuelke, Markus; Huizenga, Carin R.; Leistritz, Dru F.; Martin, George M.; Mian, I. Saira; Oshima, Junko

2007-01-01

The International Registry of Werner syndrome (www.wernersyndrome.org) has been providing molecular diagnosis of the Werner syndrome (WS) for the past decade. The present communication summarizes, from among 99 WS subjects, the spectrum of 50 distinct mutations discovered by our group and by others since the WRN gene (also called RECQL2 or REQ3) was first cloned in 1996; 25 of these have not previously been published. All WRN mutations reported thus far have resulted in the elimination of the nuclear localization signal at the C-terminus of the protein, precluding functional interactions in the nucleus; thus, all could be classified as null mutations. We now report two new mutations in the N-terminus that result in instability of the WRN protein. Clinical data confirm that the most penetrant phenotype is bilateral ocular cataracts. Other cardinal signs were seen in more than 95% of the cases. The median age of death, previously reported to be in the range of 46–48 years, is 54 years. Lymphoblastoid cell lines (LCLs) have been cryopreserved from the majority of our index cases, including material from nuclear pedigrees. These, as well as inducible and complemented hTERT (catalytic subunit of human telomerase) immortalized skin fibroblast cell lines are available to qualified investigators. Published 2006 Wiley-Liss, Inc.† PMID:16673358

Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina

PubMed Central

You, Weon-Kyoo; Bonaldo, Paolo; Stallcup, William B.

2012-01-01

To investigate the importance of the vascular basal lamina in tumor blood vessel morphogenesis and function, we compared vessel development, vessel function, and progression of B16F10 melanoma tumors in the brains of wild-type and collagen VI-null mice. In 7-day tumors in the absence of collagen VI, the width of the vascular basal lamina was reduced twofold. Although the ablation of collagen VI did not alter the abundance of blood vessels, a detailed analysis of the number of either pericytes or endothelial cells (or pericyte coverage of endothelial cells) showed that collagen VI-dependent defects during the assembly of the basal lamina have negative effects on both pericyte maturation and the sprouting and survival of endothelial cells. As a result of these deficits, vessel patency was reduced by 25%, and vessel leakiness was increased threefold, resulting in a 10-fold increase in tumor hypoxia along with a fourfold increase in hypoxia-inducible factor-1α expression. In 12-day collagen VI-null tumors, vascular endothelial growth factor expression was increased throughout the tumor stroma, in contrast to the predominantly vascular pattern of vascular endothelial growth factor expression in wild-type tumors. Vessel size was correspondingly reduced in 12-day collagen VI-null tumors. Overall, these vascular deficits produced a twofold decrease in tumor volume in collagen VI-null mice, confirming that collagen VI-dependent basal lamina assembly is a critical aspect of vessel development. PMID:22200614

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

PubMed

Jackson, Hartland W; Waterhouse, Paul; Sinha, Ankit; Kislinger, Thomas; Berman, Hal K; Khokha, Rama

2015-03-01

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.

Sf29 Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus Is a Viral Factor That Determines the Number of Virions in Occlusion Bodies▿

PubMed Central

Simón, Oihane; Williams, Trevor; Asensio, Aaron C.; Ros, Sarhay; Gaya, Andrea; Caballero, Primitivo; Possee, Robert D.

2008-01-01

The genome of Spodoptera frugiperda multiple nucleopolyhedrovirus (NPV) was inserted into a bacmid (Sfbac) and used to produce a mutant lacking open reading frame 29 (Sf29null). Sf29null bacmid DNA was able to generate an infection in S. frugiperda. Approximately six times less DNA was present in occlusion bodies (OBs) produced by the Sf29null bacmid in comparison to viruses containing this gene. This reduction in DNA content was consistent with fewer virus particles being packaged within Sf29null bacmid OBs, as determined by fractionation of dissolved polyhedra and comparison of occlusion-derived virus (ODV) infectivity in cell culture. DNA from Sfbac, Sf29null, or Sf29null-repair, in which the gene deletion had been repaired, were equally infectious when used to transfect S. frugiperda. All three viruses produced similar numbers of OBs, although those from Sf29null were 10-fold less infectious than viruses with the gene. Insects infected with Sf29null bacmid died ∼24 h later than positive controls, consistent with the reduced virus particle content of Sf29null OBs. Transcripts from Sf29 were detected in infected insects 12 h prior to those from the polyhedrin gene. Homologs to Sf29 were present in other group II NPVs, and similar sequences were present in entomopoxviruses. Analysis of the Sf29 predicted protein sequence revealed signal peptide and transmembrane domains, but the presence of 12 potential N-glycosylation sites suggest that it is not an ODV envelope protein. Other motifs, including zinc-binding and threonine-rich regions, suggest degradation and adhesion functions. We conclude that Sf29 is a viral factor that determines the number of ODVs occluded in each OB. PMID:18550678

Stable Defects in Semiconductor Nanowires.

PubMed

Sanchez, A M; Gott, J A; Fonseka, H A; Zhang, Y; Liu, H; Beanland, R

2018-05-09

Semiconductor nanowires are commonly described as being defect-free due to their ability to expel mobile defects with long-range strain fields. Here, we describe previously undiscovered topologically protected line defects with null Burgers vector that, unlike dislocations, are stable in nanoscale crystals. We analyze the defects present in semiconductor nanowires in regions of imperfect crystal growth, i.e., at the nanowire tip formed during consumption of the droplet in self-catalyzed vapor-liquid-solid growth and subsequent vapor-solid shell growth. We use a form of the Burgers circuit method that can be applied to multiply twinned material without difficulty. Our observations show that the nanowire microstructure is very different from bulk material, with line defects either (a) trapped by locks or other defects, (b) arranged as dipoles or groups with a zero total Burgers vector, or (c) have a zero Burgers vector. We find two new line defects with a null Burgers vector, formed from the combination of partial dislocations in twinned material. The most common defect is the three-monolayer high twin facet with a zero Burgers vector. Studies of individual nanowires using cathodoluminescence show that optical emission is quenched in defective regions, showing that they act as strong nonradiative recombination centers.

Btg1 is Required to Maintain the Pool of Stem and Progenitor Cells of the Dentate Gyrus and Subventricular Zone

PubMed Central

Farioli-Vecchioli, Stefano; Micheli, Laura; Saraulli, Daniele; Ceccarelli, Manuela; Cannas, Sara; Scardigli, Raffaella; Leonardi, Luca; Cinà, Irene; Costanzi, Marco; Ciotti, Maria Teresa; Moreira, Pedro; Rouault, Jean-Pierre; Cestari, Vincenzo; Tirone, Felice

2012-01-01

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal. PMID:22969701

Loss of Nrf2 promotes alveolar type 2 cell loss in irradiated, fibrotic lung.

PubMed

Traver, Geri; Mont, Stacey; Gius, David; Lawson, William E; Ding, George X; Sekhar, Konjeti R; Freeman, Michael L

2017-11-01

The development of radiation-induced pulmonary fibrosis represents a critical clinical issue limiting delivery of therapeutic doses of radiation to non-small cell lung cancer. Identification of the cell types whose injury initiates a fibrotic response and the underlying biological factors that govern that response are needed for developing strategies that prevent or mitigate fibrosis. C57BL/6 mice (wild type, Nrf2 null, Nrf2 flox/flox , and Nrf2 Δ/Δ ; SPC-Cre) were administered a thoracic dose of 12Gy and allowed to recover for 250 days. Whole slide digital and confocal microscopy imaging of H&E, Masson's trichrome and immunostaining were used to assess tissue remodeling, collagen deposition and cell renewal/mobilization during the regenerative process. Histological assessment of irradiated, fibrotic wild type lung revealed significant loss of alveolar type 2 cells 250 days after irradiation. Type 2 cell loss and the corresponding development of fibrosis were enhanced in the Nrf2 null mouse. Yet, conditional deletion of Nrf2 in alveolar type 2 cells in irradiated lung did not impair type 2 cell survival nor yield an increased fibrotic phenotype. Instead, radiation-induced ΔNp63 stem/progenitor cell mobilization was inhibited in the Nrf2 null mouse while the propensity for radiation-induced myofibroblasts derived from alveolar type 2 cells was magnified. In summary, these results indicate that Nrf2 is an important regulator of irradiated lung's capacity to maintain alveolar type 2 cells, whose injury can initiate a fibrotic phenotype. Loss of Nrf2 inhibits ΔNp63 stem/progenitor mobilization, a key event for reconstitution of injured lung, while promoting a myofibroblast phenotype that is central for fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.

PubMed

Knoop, Kathryn A; Kumar, Nachiket; Butler, Betsy R; Sakthivel, Senthilkumar K; Taylor, Rebekah T; Nochi, Tomonori; Akiba, Hisaya; Yagita, Hideo; Kiyono, Hiroshi; Williams, Ifor R

2009-11-01

Microfold cells (M cells) are specialized epithelial cells situated over Peyer's patches (PP) and other organized mucosal lymphoid tissues that transport commensal bacteria and other particulate Ags into intraepithelial pockets accessed by APCs. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL) is selectively expressed by subepithelial stromal cells in PP domes. We found that RANKL null mice have <2% of wild-type levels of PP M cells and markedly diminished uptake of 200 nm diameter fluorescent beads. Ab-mediated neutralization of RANKL in adult wild-type mice also eliminated most PP M cells. The M cell deficit in RANKL null mice was corrected by systemic administration of exogenous RANKL. Treatment with RANKL also induced the differentiation of villous M cells on all small intestinal villi with the capacity for avid uptake of Salmonella and Yersinia organisms and fluorescent beads. The RANK receptor for RANKL is expressed by epithelial cells throughout the small intestine. We conclude that availability of RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal epithelial precursor cells.

Hepatocyte growth factor enhances the inflammation-alleviating effect of umbilical cord-derived mesenchymal stromal cells in a bronchiolitis obliterans model.

PubMed

Cao, Xiao-Pei; Han, Dong-Mei; Zhao, Li; Guo, Zi-Kuan; Xiao, Feng-Jun; Zhang, Yi-Kun; Zhang, Xiao-Yan; Wang, Li-Sheng; Wang, Heng-Xiang; Wang, Hua

2016-03-01

Specific and effective therapy for prevention or reversal of bronchiolitis obliterans (BO) is lacking. In this study, we evaluated the therapeutic effect of hepatocyte growth factor (HGF) gene modified mesenchymal stromal cells (MSCs) on BO. A mouse model of experimental BO was established by subcutaneously transplanting the tracheas from C57BL/6 mice into Balb/C recipients, which were then administered saline, Ad-HGF-modified human umbilical cord-MSCs (MSCs-HGF) or Ad-Null-modified MSCs (MSCs-Null). The therapeutic effects of MSCs-Null and MSCs-HGF were evaluated by using fluorescence-activated cell sorting (FACS) for lymphocyte immunophenotype of spleen, enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (rt-PCR) for cytokine expression, and histopathological analysis for the transplanted trachea. The histopathologic recovery of allograft tracheas was improved significantly after MSCs-Null and MSCs-HGF treatment and the beneficial effects were particularly observed in MSCs-HGF-treated mice. Furthermore, the allo-transplantation-induced immunophenotype disorders of the spleen, including regulatory T (Treg), T helper (Th)1, Th2 and Th17, were attenuated in both cell-treated groups. MSCs-HGF treatment reduced expression and secretion of inflammation cytokines interferon-gamma (IFN-γ), and increased expression of anti-inflammatory cytokine interleukin (IL)-4 and IL-10. It also decreased the expression level of the profibrosis factor transforming growth factor (TGF)-β. Treatment of BO with HGF gene modified MSCs results in reduction of local inflammation and promotion in recovery of allograft trachea histopathology. These findings might provide an effective therapeutic strategy for BO. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation.

PubMed

Grasso, Francesca; Ruggieri, Vitalba; De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-04-10

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh-/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh-/- mice whereas similarly treated wild-type animals remained tumor-free.

Hhex Regulates Hematopoietic Stem Cell Self-Renewal and Stress Hematopoiesis via Repression of Cdkn2a.

PubMed

Jackson, Jacob T; Shields, Benjamin J; Shi, Wei; Di Rago, Ladina; Metcalf, Donald; Nicola, Nicos A; McCormack, Matthew P

2017-08-01

The hematopoietically expressed homeobox transcription factor (Hhex) is important for the maturation of definitive hematopoietic progenitors and B-cells during development. We have recently shown that in adult hematopoiesis, Hhex is dispensable for maintenance of hematopoietic stem cells (HSCs) and myeloid lineages but essential for the commitment of common lymphoid progenitors (CLPs) to lymphoid lineages. Here, we show that during serial bone marrow transplantation, Hhex-deleted HSCs are progressively lost, revealing an intrinsic defect in HSC self-renewal. Moreover, Hhex-deleted mice show markedly impaired hematopoietic recovery following myeloablation, due to a failure of progenitor expansion. In vitro, Hhex-null blast colonies were incapable of replating, implying a specific requirement for Hhex in immature progenitors. Transcriptome analysis of Hhex-null Lin - Sca + Kit + cells showed that Hhex deletion leads to derepression of polycomb repressive complex 2 (PRC2) and PRC1 target genes, including the Cdkn2a locus encoding the tumor suppressors p16 Ink 4 a and p19 Arf . Indeed, loss of Cdkn2a restored the capacity of Hhex-null blast colonies to generate myeloid progenitors in vitro, as well as hematopoietic reconstitution following myeloablation in vivo. Thus, HSCs require Hhex to promote PRC2-mediated Cdkn2a repression to enable continued self-renewal and response to hematopoietic stress. Stem Cells 2017;35:1948-1957. © 2017 AlphaMed Press.

New phenotypes generated by the G57R mutation of BUD23 in Saccharomyces cerevisiae.

PubMed

Lin, Jyun-Liang; Yu, Hui-Chia; Chao, Ju-Lan; Wang, Chung; Cheng, Ming-Yuan

2012-12-01

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

T cell PPAR γ is required for the anti-inflammatory efficacy of abscisic acid against experimental IBD

PubMed Central

Guri, Amir J; Evans, Nicholas P.; Hontecillas, Raquel; Bassaganya-Riera, Josep

2010-01-01

The phytohormone abscisic acid (ABA) has been shown to be effective in ameliorating chronic and acute inflammation. The objective of this study was to investigate whether ABA’s anti-inflammatory efficacy in the gut is dependent on peroxisome proliferator activated receptor γ (PPAR γ) in T cells. PPAR γ-expressing and T cell-specific PPAR γ null mice were fed diets with or without ABA (100 mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily, and mice were euthanized on day 7 of the DSS challenge. Colonic inflammation was assessed through macroscopic and histopathological examination of inflammatory lesions and real-time quantitative RT-PCR-based quantification of inflammatory genes. Flow cytometry was used to phenotypically characterize leukocyte populations in the blood and mesenteric lymph nodes (MLN). Colonic sections were stained immunohistochemically to determine the effect of ABA on colonic regulatory T (Treg) cells. ABA’s beneficial effects on disease activity were completely abrogated in T cell-specific PPAR γ null mice. Additionally, ABA improved colon histopathology, reduced blood F4/80+CD11b+ monocytes, increased the percentage of CD4+ T cells expressing the inhibitory molecule cytotoxic T lymphocyte antigen 4 (CTLA4) in blood, and enhanced the number of Treg cells in the MLN and colons of PPAR γ expressing but not T cell-specific PPAR γ null mice. We conclude that dietary ABA ameliorates experimental IBD by enhancing Treg accumulation in the colonic lamina propria through a PPAR γ-dependent mechanism. PMID:21109419

An Improved Protocol for Efficient Engraftment in NOD/LTSZ-SCIDIL-2RγNULL Mice Allows HIV Replication and Development of Anti-HIV Immune Responses

PubMed Central

Singh, Maneesh; Singh, Pratibha; Gaudray, Gilles; Musumeci, Lucia; Thielen, Caroline; Vaira, Dolores; Vandergeeten, Claire; Delacroix, Laurence; Van Gulck, Ellen; Vanham, Guido; de Leval, Laurence; Rahmouni, Souad; Moutschen, Michel

2012-01-01

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγnull (NSG) and NOD/SCID/IL2Rγnull (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3–4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials. PMID:22675567

ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

PubMed Central

Huynh, Julie M.; Dang, Hope; Munoz-Tucker, Isabel A.; O’Ketch, Marvin; Liu, Ian T.; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

2016-01-01

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5. PMID:26596346

Kv7.2 regulates the function of peripheral sensory neurons.

PubMed

King, Chih H; Lancaster, Eric; Salomon, Daniela; Peles, Elior; Scherer, Steven S

2014-10-01

The Kv7 (KCNQ) family of voltage-gated K(+) channels regulates cellular excitability. The functional role of Kv7.2 has been hampered by the lack of a viable Kcnq2-null animal model. In this study, we generated homozygous Kcnq2-null sensory neurons using the Cre-Lox system; in these mice, Kv7.2 expression is absent in the peripheral sensory neurons, whereas the expression of other molecular components of nodes (including Kv7.3), paranodes, and juxtaparanodes is not altered. The conditional Kcnq2-null animals exhibit normal motor performance but have increased thermal hyperalgesia and mechanical allodynia. Whole-cell patch recording technique demonstrates that Kcnq2-null sensory neurons have increased excitability and reduced spike frequency adaptation. Taken together, our results suggest that the loss of Kv7.2 activity increases the excitability of primary sensory neurons. © 2014 Wiley Periodicals, Inc.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via Insulin-like growth factor-1 receptor-independent mechanism

PubMed Central

Waraky, Ahmed; Akopyan, Karen; Parrow, Vendela; Strömberg, Thomas; Axelson, Magnus; Abrahmsén, Lars; Lindqvist, Arne; Larsson, Olle; Aleem, Eiman

2014-01-01

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP. PMID:25268741

Magnetohydrodynamic Modeling of Solar Coronal Dynamics with an Initial Non-force-free Magnetic Field

NASA Astrophysics Data System (ADS)

Prasad, A.; Bhattacharyya, R.; Kumar, Sanjay

2017-05-01

The magnetic fields in the solar corona are generally neither force-free nor axisymmetric and have complex dynamics that are difficult to characterize. Here we simulate the topological evolution of solar coronal magnetic field lines (MFLs) using a magnetohydrodynamic model. The simulation is initialized with a non-axisymmetric non-force-free magnetic field that best correlates with the observed vector magnetograms of solar active regions (ARs). To focus on these ideas, simulations are performed for the flaring AR 11283 noted for its complexity and well-documented dynamics. The simulated dynamics develops as the initial Lorentz force pushes the plasma and facilitates successive magnetic reconnections at the two X-type null lines present in the initial field. Importantly, the simulation allows for the spontaneous development of mass flow, unique among contemporary works, that preferentially reconnects field lines at one of the X-type null lines. Consequently, a flux rope consisting of low-lying twisted MFLs, which approximately traces the major polarity inversion line, undergoes an asymmetric monotonic rise. The rise is attributed to a reduction in the magnetic tension force at the region overlying the rope, resulting from the reconnection. A monotonic rise of the rope is in conformity with the standard scenario of flares. Importantly, the simulated dynamics leads to bifurcations of the flux rope, which, being akin to the observed filament bifurcation in AR 11283, establishes the appropriateness of the initial field in describing ARs.

Magnetohydrodynamic Modeling of Solar Coronal Dynamics with an Initial Non-force-free Magnetic Field

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

Prasad, A.; Bhattacharyya, R.; Kumar, Sanjay

The magnetic fields in the solar corona are generally neither force-free nor axisymmetric and have complex dynamics that are difficult to characterize. Here we simulate the topological evolution of solar coronal magnetic field lines (MFLs) using a magnetohydrodynamic model. The simulation is initialized with a non-axisymmetric non-force-free magnetic field that best correlates with the observed vector magnetograms of solar active regions (ARs). To focus on these ideas, simulations are performed for the flaring AR 11283 noted for its complexity and well-documented dynamics. The simulated dynamics develops as the initial Lorentz force pushes the plasma and facilitates successive magnetic reconnections atmore » the two X-type null lines present in the initial field. Importantly, the simulation allows for the spontaneous development of mass flow, unique among contemporary works, that preferentially reconnects field lines at one of the X-type null lines. Consequently, a flux rope consisting of low-lying twisted MFLs, which approximately traces the major polarity inversion line, undergoes an asymmetric monotonic rise. The rise is attributed to a reduction in the magnetic tension force at the region overlying the rope, resulting from the reconnection. A monotonic rise of the rope is in conformity with the standard scenario of flares. Importantly, the simulated dynamics leads to bifurcations of the flux rope, which, being akin to the observed filament bifurcation in AR 11283, establishes the appropriateness of the initial field in describing ARs.« less

Impaired angiogenesis in aminopeptidase N-null mice

PubMed Central

Rangel, Roberto; Sun, Yan; Guzman-Rojas, Liliana; Ozawa, Michael G.; Sun, Jessica; Giordano, Ricardo J.; Van Pelt, Carolyn S.; Tinkey, Peggy T.; Behringer, Richard R.; Sidman, Richard L.; Arap, Wadih; Pasqualini, Renata

2007-01-01

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases. PMID:17360568

Control of recollection by slow gamma dominating mid-frequency gamma in hippocampus CA1

PubMed Central

Dvorak, Dino; Radwan, Basma; Sparks, Fraser T.; Talbot, Zoe Nicole

2018-01-01

Behavior is used to assess memory and cognitive deficits in animals like Fmr1-null mice that model Fragile X Syndrome, but behavior is a proxy for unknown neural events that define cognitive variables like recollection. We identified an electrophysiological signature of recollection in mouse dorsal Cornu Ammonis 1 (CA1) hippocampus. During a shocked-place avoidance task, slow gamma (SG) (30–50 Hz) dominates mid-frequency gamma (MG) (70–90 Hz) oscillations 2–3 s before successful avoidance, but not failures. Wild-type (WT) but not Fmr1-null mice rapidly adapt to relocating the shock; concurrently, SG/MG maxima (SGdom) decrease in WT but not in cognitively inflexible Fmr1-null mice. During SGdom, putative pyramidal cell ensembles represent distant locations; during place avoidance, these are avoided places. During shock relocation, WT ensembles represent distant locations near the currently correct shock zone, but Fmr1-null ensembles represent the formerly correct zone. These findings indicate that recollection occurs when CA1 SG dominates MG and that accurate recollection of inappropriate memories explains Fmr1-null cognitive inflexibility. PMID:29346381

Expression of the voltage-gated potassium channel KCNQ1 in mammalian taste bud cells and the effect of its null-mutation on taste preferences.

PubMed

Wang, Hong; Iguchi, Naoko; Rong, Qi; Zhou, Minliang; Ogunkorode, Martina; Inoue, Masashi; Pribitkin, Edmund A; Bachmanov, Alexander A; Margolskee, Robert F; Pfeifer, Karl; Huang, Liquan

2009-01-20

Vertebrate taste buds undergo continual cell turnover. To understand how the gustatory progenitor cells in the stratified lingual epithelium migrate and differentiate into different types of mature taste cells, we sought to identify genes that were selectively expressed in taste cells at different maturation stages. Here we report the expression of the voltage-gated potassium channel KCNQ1 in mammalian taste buds of mouse, rat, and human. Immunohistochemistry and nuclear staining showed that nearly all rodent and human taste cells express this channel. Double immunostaining with antibodies against type II and III taste cell markers validated the presence of KCNQ1 in these two types of cells. Co-localization studies with cytokeratin 14 indicated that KCNQ1 is also expressed in type IV basal precursor cells. Null mutation of the kcnq1 gene in mouse, however, did not alter the gross structure of taste buds or the expression of taste signaling molecules. Behavioral assays showed that the mutant mice display reduced preference to some umami substances, but not to any other taste compounds tested. Gustatory nerve recordings, however, were unable to detect any significant change in the integrated nerve responses of the mutant mice to umami stimuli. These results suggest that although it is expressed in nearly all taste bud cells, the function of KCNQ1 is not required for gross taste bud development or peripheral taste transduction pathways, and the reduced preference of kcnq1-null mice in the behavioral assays may be attributable to the deficiency in the central nervous system or other organs.

PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons.

PubMed

Wang, Liheng; Sui, Lina; Panigrahi, Sunil K; Meece, Kana; Xin, Yurong; Kim, Jinrang; Gromada, Jesper; Doege, Claudia A; Wardlaw, Sharon L; Egli, Dieter; Leibel, Rudolph L

2017-02-14

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Deletion of SLC19A2, the high affinity thiamine transporter, causes selective inner hair cell loss and an auditory neuropathy phenotype.

PubMed

Liberman, M C; Tartaglini, E; Fleming, J C; Neufeld, E J

2006-09-01

Mutations in the gene coding for the high-affinity thiamine transporter Slc19a2 underlie the clinical syndrome known as thiamine-responsive megaloblastic anemia (TRMA) characterized by anemia, diabetes, and sensorineural hearing loss. To create a mouse model of this disease, a mutant line was created with targeted disruption of the gene. Cochlear function is normal in these mutants when maintained on a high-thiamine diet. When challenged with a low-thiamine diet, Slc19a2-null mice showed 40-60 dB threshold elevations by auditory brainstem response (ABR), but only 10-20 dB elevation by otoacoustic emission (OAE) measures. Wild-type mice retain normal hearing on either diet. Cochlear histological analysis showed a pattern uncommon for sensorineural hearing loss: selective loss of inner hair cells after 1-2 weeks on low thiamine and significantly greater inner than outer hair cell loss after longer low-thiamine challenges. Such a pattern is consistent with the observed discrepancy between ABR and OAE threshold shifts. The possible role of thiamine transport in other reported cases of selective inner hair cell loss is considered.

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

PubMed

Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

2013-08-23

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins

PubMed Central

Stephens, Andrew D.; Liu, Patrick Z.; Banigan, Edward J.; Almassalha, Luay M.; Backman, Vadim; Adam, Stephen A.; Goldman, Robert D.; Marko, John F.

2018-01-01

Nuclear shape and architecture influence gene localization, mechanotransduction, transcription, and cell function. Abnormal nuclear morphology and protrusions termed “blebs” are diagnostic markers for many human afflictions including heart disease, aging, progeria, and cancer. Nuclear blebs are associated with both lamin and chromatin alterations. A number of prior studies suggest that lamins dictate nuclear morphology, but the contributions of altered chromatin compaction remain unclear. We show that chromatin histone modification state dictates nuclear rigidity, and modulating it is sufficient to both induce and suppress nuclear blebs. Treatment of mammalian cells with histone deacetylase inhibitors to increase euchromatin or histone methyltransferase inhibitors to decrease heterochromatin results in a softer nucleus and nuclear blebbing, without perturbing lamins. Conversely, treatment with histone demethylase inhibitors increases heterochromatin and chromatin nuclear rigidity, which results in reduced nuclear blebbing in lamin B1 null nuclei. Notably, increased heterochromatin also rescues nuclear morphology in a model cell line for the accelerated aging disease Hutchinson–Gilford progeria syndrome caused by mutant lamin A, as well as cells from patients with the disease. Thus, chromatin histone modification state is a major determinant of nuclear blebbing and morphology via its contribution to nuclear rigidity. PMID:29142071

The lateral mobility of cell adhesion molecules is highly restricted at septate junctions in Drosophila.

PubMed

Laval, Monique; Bel, Christophe; Faivre-Sarrailh, Catherine

2008-07-18

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions. We conducted photobleaching experiments on whole living Drosophila embryos to analyze the membrane mobility of CAMs at septate junctions between epithelial cells. We show that GFP-tagged Nrg and Nrx IV molecules exhibit very stable association with septate junctions in wild-type embryos. Nrg-GFP is mislocalized to the baso-lateral membrane in nrx IV or cont null mutant embryos, and displays increased mobile fraction. Similarly, Nrx IV-GFP becomes distributed to the baso-lateral membrane in null mutants of vari and cora, and its mobile fraction is strongly increased. The loss of Vari, a MAGUK protein that interacts with the cytoplasmic tail of Nrx IV, has a stronger effect than the null mutation of nrx IV on the lateral mobility of Nrg-GFP. The strands of septate junctions display a stable behavior in vivo that may be correlated with their role of paracellular barrier. The membrane mobility of CAMs is strongly limited when they take part to the multimolecular complex forming septate junctions. This restricted lateral diffusion of CAMs depends on both adhesive interactions and clustering by scaffolding molecules. The lateral mobility of CAMs is strongly increased in embryos presenting alteration of septate junctions. The stronger effect of vari by comparison with nrx IV null mutation supports the hypothesis that this scaffolding molecule may cross-link different types of CAMs and play a crucial role in stabilizing the strands of septate junctions.

Keratinocyte p38δ loss inhibits Ras-induced tumor formation, while systemic p38δ loss enhances skin inflammation in the early phase of chemical carcinogenesis in mouse skin.

PubMed

Kiss, Alexi; Koppel, Aaron C; Anders, Joanna; Cataisson, Christophe; Yuspa, Stuart H; Blumenberg, Miroslav; Efimova, Tatiana

2016-05-01

p38δ expression and/or activity are increased in human cutaneous malignancies, including invasive squamous cell carcinoma (SCC) and head and neck SCC, but the role of p38δ in cutaneous carcinogenesis has not been well-defined. We have reported that mice with germline loss of p38δ exhibited a reduced susceptibility to skin tumor development compared with wild-type mice in the two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical skin carcinogenesis model. Here, we report that p38δ gene ablation inhibited the growth of tumors generated from v-ras(Ha) -transformed keratinocytes in skin orthografts to nude mice, indicating that keratinocyte-intrinsic p38δ is required for Ras-induced tumorigenesis. Gene expression profiling of v-ras(Ha) -transformed p38δ-null keratinocytes revealed transcriptional changes associated with cellular responses linked to tumor suppression, such as reduced proliferation and increased differentiation, cell adhesion, and cell communications. Notably, a short-term DMBA/TPA challenge, modeling the initial stages of chemical skin carcinogenesis treatment, elicited an enhanced inflammation in p38δ-null skin compared with skin of wild-type mice, as assessed by measuring the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNFα. Additionally, p38δ-null skin and p38δ-null keratinocytes exhibited increased p38α activation and signaling in response to acute inflammatory challenges, suggesting a role for p38α in stimulating the elevated inflammatory response in p38δ-null skin during the initial phases of the DMBA/TPA treatment compared with similarly treated p38δ(+/+) skin. Altogether, our results indicate that p38δ signaling regulates skin carcinogenesis not only by keratinocyte cell-autonomous mechanisms, but also by influencing the interaction between between the epithelial compartment of the developing skin tumor and its stromal microenvironment. © 2015 Wiley Periodicals, Inc.

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone.

PubMed

Gould, Elizabeth A; Busquet, Nicolas; Shepherd, Douglas; Dietz, Robert M; Herson, Paco S; Simoes de Souza, Fabio M; Li, Anan; George, Nicholas M; Restrepo, Diego; Macklin, Wendy B

2018-02-13

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1 -null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1 -null mice. Young adult Plp1- null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption. © 2018, Gould et al.

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone

PubMed Central

Gould, Elizabeth A; Busquet, Nicolas; Shepherd, Douglas; Dietz, Robert M; Herson, Paco S; Simoes de Souza, Fabio M; Li, Anan; George, Nicholas M

2018-01-01

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption. PMID:29436368

Reconnection at three dimensional magnetic null points: Effect of current sheet asymmetry

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

Wyper, P. F.; Jain, Rekha

2013-05-15

Asymmetric current sheets are likely to be prevalent in both astrophysical and laboratory plasmas with complex three dimensional (3D) magnetic topologies. This work presents kinematic analytical models for spine and fan reconnection at a radially symmetric 3D null (i.e., a null where the eigenvalues associated with the fan plane are equal) with asymmetric current sheets. Asymmetric fan reconnection is characterized by an asymmetric reconnection of flux past each spine line and a bulk flow of plasma across the null point. In contrast, asymmetric spine reconnection is characterized by the reconnection of an equal quantity of flux across the fan planemore » in both directions. The higher modes of spine reconnection also include localized wedges of vortical flux transport in each half of the fan. In this situation, two definitions for reconnection rate become appropriate: a local reconnection rate quantifying how much flux is genuinely reconnected across the fan plane and a global rate associated with the net flux driven across each semi-plane. Through a scaling analysis, it is shown that when the ohmic dissipation in the layer is assumed to be constant, the increase in the local rate bleeds from the global rate as the sheet deformation is increased. Both models suggest that asymmetry in the current sheet dimensions will have a profound effect on the reconnection rate and manner of flux transport in reconnection involving 3D nulls.« less

Identifying the null subject: evidence from event-related brain potentials.

PubMed

Demestre, J; Meltzer, S; García-Albea, J E; Vigil, A

1999-05-01

Event-related brain potentials (ERPs) were recorded during spoken language comprehension to study the on-line effects of gender agreement violations in controlled infinitival complements. Spanish sentences were constructed in which the complement clause contained a predicate adjective marked for syntactic gender. By manipulating the gender of the antecedent (i.e., the controller) of the implicit subject while holding constant the gender of the adjective, pairs of grammatical and ungrammatical sentences were created. The detection of such a gender agreement violation would indicate that the parser had established the coreference relation between the null subject and its antecedent. The results showed a complex biphasic ERP (i.e., an early negativity with prominence at anterior and central sites, followed by a centroparietal positivity) in the violating condition as compared to the non-violating conditions. The brain reacts to NP-adjective gender agreement violations within a few hundred milliseconds of their occurrence. The data imply that the parser has properly coindexed the null subject of an infinitive clause with its antecedent.

Simple Map with Low MN Perturbation for a Single-Null Divertor Tokamak with Constant Width of Stochastic Layer

NASA Astrophysics Data System (ADS)

Verma, Arun; Smith, Terry; Punjabi, Alkesh; Boozer, Allen

1996-11-01

In this work, we investigate the effects of low MN perturbations in a single-null divertor tokamak with stochastic scrape-off layer. The unperturbed magnetic topology of a single-null divertor tokamak is represented by Simple Map (Punjabi A, Verma A and Boozer A, Phys Rev Lett), 69, 3322 (1992) and J Plasma Phys, 52, 91 (1994). We choose the combinations of the map parameter k, and the strength of the low MN perturbation such that the width of stochastic layer remains unchanged. We give detailed results on the effects of low MN perturbation on the magnetic topology of the stochastic layer and on the footprint of field lines on the divertor plate given the constraint of constant width of the stochastic layer. The low MN perturbations occur naturally and therefore their effects are of considerable importance in tokamak divertor physics. This work is supported by US DOE OFES. Use of CRAY at HU and at NERSC is gratefully acknowledged.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

PubMed

Guzman-Rojas, Liliana; Rangel, Roberto; Salameh, Ahmad; Edwards, Julianna K; Dondossola, Eleonora; Kim, Yun-Gon; Saghatelian, Alan; Giordano, Ricardo J; Kolonin, Mikhail G; Staquicini, Fernanda I; Koivunen, Erkki; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2012-01-31

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

The Cytoplasmic C-Tail of the Mouse Cytomegalovirus 7 Transmembrane Receptor Homologue, M78, Regulates Endocytosis of the Receptor and Modulates Virus Replication in Different Cell Types

PubMed Central

2016-01-01

Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes. PMID:27760189

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

PubMed

Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

2016-04-01

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Deficiency in Monocarboxylate Transporter 1 (MCT1) in Mice Delays Regeneration of Peripheral Nerves following Sciatic Nerve Crush

PubMed Central

Morrison, Brett M.; Tsingalia, Akivaga; Vidensky, Svetlana; Lee, Youngjin; Jin, Lin; Farah, Mohamed H.; Lengacher, Sylvain; Magistretti, Pierre J.; Pellerin, Luc; Rothstein, Jeffrey D.

2014-01-01

Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence and MCT1 tdTomato BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves in MCT1 heterozygous null mice are crushed and peripheral nerve regeneration quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21 days in wild-type mice to greater than 38 days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42 days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42 days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4 weeks and tibial mixed sensory and motor nerve at 3 weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly through failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush. PMID:25447940

Deficiency in monocarboxylate transporter 1 (MCT1) in mice delays regeneration of peripheral nerves following sciatic nerve crush.

PubMed

Morrison, Brett M; Tsingalia, Akivaga; Vidensky, Svetlana; Lee, Youngjin; Jin, Lin; Farah, Mohamed H; Lengacher, Sylvain; Magistretti, Pierre J; Pellerin, Luc; Rothstein, Jeffrey D

2015-01-01

Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence in wild-type mice and tdTomato fluorescence in MCT1 BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves of MCT1 heterozygous null mice are crushed and peripheral nerve regeneration was quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21 days in wild-type mice to greater than 38 days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42 days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42 days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4 weeks and tibial mixed sensory and motor nerve at 3 weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly due to failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush. Copyright © 2014 Elsevier Inc. All rights reserved.

Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

PubMed Central

Martínez-Sanz, Elena; Del Río, Aurora; Barrio, Carmen; Murillo, Jorge; Maldonado, Estela; Garcillán, Beatriz; Amorós, María; Fuerte, Tamara; Fernández, Álvaro; Trinidad, Eva; Rabadán, M Ángeles; López, Yamila; Martínez, M Luisa; Martínez-Álvarez, Concepción

2008-01-01

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice. PMID:18431835

Nrf2 activation prevents cadmium-induced acute liver injury

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

Wu, Kai C.; Liu, Jie J.; Klaassen, Curtis D., E-mail: cklaasse@kumc.edu

2012-08-15

Oxidative stress plays an important role in cadmium-induced liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the role of Nrf2 in cadmium-induced hepatotoxicity, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation were treated with cadmium chloride (3.5 mg Cd/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Cadmium increased serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities, and caused extensive hepatic hemorrhage and necrosis in the Nrf2-nullmore » mice. In contrast, Nrf2-enhanced mice had lower serum ALT and LDH activities and less morphological alternations in the livers than wild-type mice. H{sub 2}DCFDA (2′,7′-dichlorodihydrofluoresein diacetate) staining of primary hepatocytes isolated from the four genotypes of mice indicated that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. To further investigate the mechanism of the protective effect of Nrf2, mRNA of metallothionein (MT) and other cytoprotective genes were determined. Cadmium markedly induced MT-1 and MT-2 in livers of all four genotypes of mice. In contrast, genes involved in glutathione synthesis and reducing reactive oxygen species, including glutamate-cysteine ligase (Gclc), glutathione peroxidase-2 (Gpx2), and sulfiredoxin-1 (Srxn-1) were only induced in Nrf2-enhanced mice, but not in Nrf2-null mice. In conclusion, the present study shows that Nrf2 activation prevents cadmium-induced oxidative stress and liver injury through induction of genes involved in antioxidant defense rather than genes that scavenge Cd. -- Highlights: ► Cadmium caused extensive hepatic hemorrhage and necrosis in Nrf2-null mice. ► Keap1-KD and Keap1-HKO mice were resistant to cadmium-induced liver injury. ► Cadmium increased ROS in hepatocytes isolated from Nrf2-null and wild-type mice. ► Mt-1 and Mt‐2 were induced over 200-fold in both Nrf2-null and Nrf2-enhanced mice. ► Gclc, Gpx2, and Srxn-1 were induced in Nrf2-enhanced mice, not in Nrf2-null mice.« less

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus.

PubMed

Deschamps, Thibaut; Kalamvoki, Maria

2017-05-01

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1. Copyright © 2017 American Society for Microbiology.

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

PubMed Central

Deschamps, Thibaut

2017-01-01

ABSTRACT Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2′3′-cyclic GAMP (2′3′-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1. PMID:28179534

Modulation of glucocorticoid resistance in pediatric T-cell Acute Lymphoblastic Leukemia by increasing BIM expression with the PI3K/mTOR inhibitor BEZ235

PubMed Central

Hall, Connor P.; Reynolds, C. Patrick; Kang, Min H.

2015-01-01

Purpose The aim of our study is to evaluate the preclinical therapeutic activity and mechanism of action of BEZ235, a dual PI3K/mTOR inhibitor, in combination with dexamethasone in ALL. Experimental Design The cytotoxic effects of BEZ235 and dexamethasone as single agents and in combination were assessed in a panel of ALL cell lines and xenograft models. The underlying mechanism of BEZ235 and dexamethasone was evaluated using immunoblotting, TaqMan RT-PCR, siRNA, immunohistochemistry and immunoprecipitation. Results Inhibition of the PI3K/AKT/mTOR pathway with the dual PI3K/mTOR inhibitor BEZ235 enhanced dexamethasone-induced anti-leukemic activity in in vitro (continuous cell lines and primary ALL cultures) and systemic in vivo models of T-ALL (including a patient-derived xenograft). Through inhibition of AKT1, BEZ235 was able to alleviate AKT1-mediated suppression of dexamethasone-induced apoptotic pathways leading to increased expression of the pro-apoptotic BCL-2 protein BIM. Downregulation of MCL-1 by BEZ235 further contributed to the modulation of dexamethasone resistance by increasing the amount of BIM available to induce apoptosis, especially in PTEN-null T-ALL where inhibition of AKT only partially overcame AKT-induced BIM suppression. Conclusion Our data support the further investigation of agents targeting the PI3K/mTOR pathway to modulate glucocorticoid resistance in T-ALL. PMID:26080839

Testing natural selection vs. genetic drift in phenotypic evolution using quantitative trait locus data.

PubMed Central

Orr, H A

1998-01-01

Evolutionary biologists have long sought a way to determine whether a phenotypic difference between two taxa was caused by natural selection or random genetic drift. Here I argue that data from quantitative trait locus (QTL) analyses can be used to test the null hypothesis of neutral phenotypic evolution. I propose a sign test that compares the observed number of plus and minus alleles in the "high line" with that expected under neutrality, conditioning on the known phenotypic difference between the taxa. Rejection of the null hypothesis implies a role for directional natural selection. This test is applicable to any character in any organism in which QTL analysis can be performed. PMID:9691061

The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types.

PubMed

Hersh, Bradley M; Hartwieg, Erika; Horvitz, H Robert

2002-04-02

The misregulation of programmed cell death, or apoptosis, contributes to the pathogenesis of many diseases. We used Nomarski microscopy to screen for mutants containing refractile cell corpses in a C. elegans strain in which all programmed cell death is blocked and such corpses are absent. We isolated a mutant strain that accumulates refractile bodies resembling irregular cell corpses. We rescued this mutant phenotype with the C. elegans mucolipidosis type IV (ML-IV) homolog, the recently identified cup-5 (coelomocyte-uptake defective) gene. ML-IV is a human autosomal recessive lysosomal storage disease characterized by psychomotor retardation and ophthalmological abnormalities. Our null mutations in cup-5 cause maternal-effect lethality. In addition, cup-5 mutants contain excess lysosomes in many and possibly all cell types and contain lamellar structures similar to those observed in ML-IV cell lines. The human ML-IV gene is capable of rescuing both the maternal-effect lethality and the lysosome-accumulation abnormality of cup-5 mutants. cup-5 mutants seem to contain excess apoptotic cells as detected by staining with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. We suggest that the increased apoptosis seen in cup-5 mutants is a secondary consequence of the lysosomal defect, and that abnormalities in apoptosis may be associated with human lysosomal storage disorders.

The anthracenedione compound bostrycin induces mitochondria-mediated apoptosis in the yeast Saccharomyces cerevisiae.

PubMed

Xu, Chunling; Wang, Jiafeng; Gao, Ye; Lin, Huangyu; Du, Lin; Yang, Shanshan; Long, Simei; She, Zhigang; Cai, Xiaoling; Zhou, Shining; Lu, Yongjun

2010-05-01

Bostrycin is an anthracenedione with phytotoxic and antibacterial activity that belongs to the large family of quinones. We have isolated bostrycin from the secondary metabolites of a mangrove endophytic fungus, no. 1403, collected from the South China Sea. Using the yeast Saccharomyces cerevisiae as a model, we show that bostrycin inhibits cell proliferation by blocking the cell cycle at G1 phase and ultimately leads to cell death in a time- and dose-dependent manner. Bostrycin-induced lethal cytotoxicity is accompanied with increased levels of intracellular reactive oxygen species and hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and externalization of phosphatidylserine. We further show that bostrycin decreases mitochondrial membrane electric potential and causes mitochondrial destruction during the progression of cell death. Bostrycin-induced cell death was promoted in YCA1 null yeast strain but was partially rescued in AIF1 null mutant both in fermentative and respiratory media, strongly indicating that bostrycin induces apoptosis in yeast cells through a mitochondria-mediated but caspase-independent pathway.

Candida albicans Iff11, a secreted protein required for cell wall structure and virulence.

PubMed

Bates, Steven; de la Rosa, José M; MacCallum, Donna M; Brown, Alistair J P; Gow, Neil A R; Odds, Frank C

2007-06-01

The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be O glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

Identifying Neurofibromin-Specific Regulatory Nodes for Therapeutic Targeting in NF1

DTIC Science & Technology

2016-10-01

adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology to find genes that regulate...neurofibromin depends on the adapter protein SPRED1, to function, and we are utilizing the latest technical innovations including CRISPR technology...signaling NF1-Null HEK 293T cells have been generated using CRISPR /Cas9 and single clones have been expanded for biochemical assays (Figure 2). NF1-Null

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

PubMed

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Role of a new Rho family member in cell migration and axon guidance in C. elegans.

PubMed

Zipkin, I D; Kindt, R M; Kenyon, C J

1997-09-05

Rho family GTPases are thought to regulate actin-dependent processes, but their functions in vivo are still poorly understood. We have investigated the function of a new, widely expressed Rho family member in C. elegans by analyzing mutations in the endogenous gene. Activated and null alleles all inhibit cell migration, demonstrating that this protein is required for cell migration in vivo. Only a small subset of the migrations inhibited by activating mutations are inhibited by null mutations, suggesting that considerable functional redundancy exists within this system. Our findings support this conclusion and show that mig-2 functions redundantly with another pathway to regulate nuclear migration. Surprisingly, activated alleles also cause misguided axon growth, suggesting that Rho family GTPases may couple guidance cues to process outgrowth.

Reversing hypomyelination in BACE1-null mice with Akt-DD overexpression.

PubMed

Hu, Xiangyou; Schlanger, Rita; He, Wanxia; Macklin, Wendy B; Yan, Riqiang

2013-05-01

β-Site amyloid precursor protein convertase enzyme 1 (BACE1), a type I transmembrane aspartyl protease required to cleave amyloid precursor protein for releasing a toxic amyloid peptide, also cleaves type I and type III neuregulin-1 (Nrg-1). BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination if injured. In BACE1-null mice, the abolished cleavage of neuregulin-1 by BACE1 is speculated to cause reduced myelin sheath thickness in both the central nervous system and peripheral nervous system because reduced cleavage of Nrg-1 correlates with reduced Akt phosphorylation, a downstream signaling molecule of the Nrg-1/ErbB pathway. Here we tested specifically whether increasing Akt activity alone in oligodendrocytes would be sufficient to reverse the hypomyelination phenotype in BACE1-null mice. BACE1-null mice were bred with transgenic mice expressing constitutively active Akt (Akt-DD; mutations with D(308)T and D(473)S) in oligodendrocytes. Relative to littermate BACE1-null controls, BACE1(-/-)/Akt-DD mice exhibited enhanced expression of myelin basic protein and promoter of proteolipid protein. The elevated expression of myelin proteins correlated with a thicker myelin sheath in optic nerves; comparison of quantified g ratios with statistic significance was used to confirm this reversion. However, it appeared that myelin sheath thickness in the sciatic nerves was not increased in BACE1(-/-)/Akt-DD mice, as the g ratio was not significantly different from the control. Hence, increased Akt activity in BACE1-null myelinating cells only compensates for the loss of BACE1 activity in the central nervous system, which is consistent with the observation that overexpression of Akt-DD in Schwann cells did not induce hypermyelination. Our results suggest that signaling activity other than Akt may also contribute to proper myelination in peripheral nerves.

Panobinostat Enhances Cytarabine and Daunorubicin Sensitivities in AML Cells through Suppressing the Expression of BRCA1, CHK1, and Rad51

PubMed Central

Edwards, Holly; Caldwell, J. Timothy; Chen, Wei; Inaba, Hiroto; Xu, Xuelian; Buck, Steven A.; Taub, Jeffrey W.; Baker, Sharyn D.; Ge, Yubin

2013-01-01

Acute myeloid leukemia (AML) remains a challenging disease to treat and urgently requires new therapies to improve its treatment outcome. In this study, we investigated the molecular mechanisms underlying the cooperative antileukemic activities of panobinostat and cytarabine or daunorubicin (DNR) in AML cell lines and diagnostic blast samples in vitro and in vivo. Panobinostat suppressed expression of BRCA1, CHK1, and RAD51 in AML cells in a dose-dependent manner. Further, panobinostat significantly increased cytarabine- or DNR-induced DNA double-strand breaks and apoptosis, and abrogated S and/or G2/M cell cycle checkpoints. Analogous results were obtained by shRNA knockdown of BRCA1, CHK1, or RAD51. Cotreatment of NOD-SCID-IL2Rγnull mice bearing AML xenografts with panobinostat and cytarabine significantly increased survival compared to either cytarabine or panobinostat treatment alone. Additional studies revealed that panobinostat suppressed the expression of BRCA1, CHK1, and RAD51 through downregulation of E2F1 transcription factor. Our results establish a novel mechanism underlying the cooperative antileukemic activities of these drug combinations in which panobinostat suppresses expression of BRCA1, CHK1, and RAD51 to enhance cytarabine and daunorubicin sensitivities in AML cells. PMID:24244429

Synergistic inhibition of the APC/C by the removal of APC15 in HCT116 cells lacking UBE2C.

PubMed

Garvanska, Dimitriya H; Larsen, Marie Sofie Yoo; Nilsson, Jakob

2016-10-15

The spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly, in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components. © 2016. Published by The Company of Biologists Ltd.

Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

PubMed Central

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

A symplectic map for trajectories of magnetic field lines in double-null divertor tokamaks

NASA Astrophysics Data System (ADS)

Crank, Willie; Ali, Halima; Punjabi, Alkesh

2009-11-01

The coordinates of the area-preserving map equations for integration of magnetic field line trajectories in tokamaks can be any coordinates for which a transformation to (ψ,θ,φ) coordinates exists [A. Punjabi, H. Ali, T. Evans, and A. Boozer, Phys. Lett. A 364, 140 (2007)]. ψ is toroidal magnetic flux, θ is poloidal angle, and φ is toroidal angle. This freedom is exploited to construct a map that represents the magnetic topology of double-null divertor tokamaks. For this purpose, the generating function of the simple map [A. Punjabi, A. Verma, and A. Boozer, Phys. Rev. Lett. 69, 3322 (1992)] is slightly modified. The resulting map equations for the double-null divertor tokamaks are: x1=x0-ky0(1-y0^2 ), y1=y0+kx1. k is the map parameter. It represents the generic topological effects of toroidal asymmetries. The O-point is at (0.0). The X-points are at (0,±1). The equilibrium magnetic surfaces are calculated. These surfaces are symmetric about the x- and y- axes. The widths of stochastic layer near the X-points in the principal plane, and the fractal dimensions of the magnetic footprints on the inboard and outboard side of upper and lower X-points are calculated from the map. This work is supported by US Department of Energy grants DE-FG02-07ER54937, DE-FG02-01ER54624 and DE-FG02-04ER54793.

Keratin 17 null mice exhibit age- and strain-dependent alopecia.

PubMed

McGowan, Kevin M; Tong, Xuemei; Colucci-Guyon, Emma; Langa, Francina; Babinet, Charles; Coulombe, Pierre A

2002-06-01

Onset of type I keratin 17 (K17) synthesis marks the adoption of an appendageal fate within embryonic ectoderm, and its expression persists in specific cell types within mature hair, glands, and nail. We report that K17 null mice develop severe alopecia during the first week postbirth, correlating with hair fragility, alterations in follicular histology, and apoptosis in matrix cells. These alterations are incompletely penetrant and normalize starting with the first postnatal cycle. Absence of a hair phenotype correlates with a genetic strain-dependent compensation by related keratins, including K16. These findings reveal a crucial role for K17 in the structural integrity of the first hair produced and the survival of hair-producing cells. Given that identical inherited mutations in this gene can cause either pachyonychia congenita or steatocystoma multiplex, the features of this mouse model suggest that this clinical heterogeneity arises from a cell type-specific, genetically determined compensation by related keratins.

The Impact of ExoS on Pseudomonas aeruginosa Internalization by Epithelial Cells Is Independent of fleQ and Correlates with Bistability of Type Three Secretion System Gene Expression.

PubMed

Kroken, Abby R; Chen, Camille K; Evans, David J; Yahr, Timothy L; Fleiszig, Suzanne M J

2018-05-01

Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. aeruginosa and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null PA103 remained less well internalized than PAO1 matched for fleQ status, but only with ExoS expression, suggesting additional differences between these strains. Quantifying T3SS activation using GFP fluorescence and quantitative reverse transcription-PCR (qRT-PCR) showed that T3SS expression was hyperinducible for strain PA103Δ exoUT versus other isolates and was unrelated to fleQ status. These findings support the principle that P. aeruginosa is not exclusively an extracellular pathogen, with internalization influenced by the relative proportions of T3SS-positive and T3SS-negative bacteria in the population during host cell interaction. These data also challenge current thinking about T3SS effector delivery into host cells and suggest that T3SS bistability is an important consideration in studying P. aeruginosa pathogenesis. IMPORTANCE P. aeruginosa is often referred to as an extracellular pathogen, despite its demonstrated capacity to invade and survive within host cells. Fueling the confusion, P. aeruginosa encodes T3SS effectors with anti-internalization activity that, paradoxically, play critical roles in intracellular survival. Here, we sought to address why ExoS does not prevent internalization of the P. aeruginosa strains that natively encode it. Results showed that ExoS exerted unusually strong anti-internalization activity under conditions of expression in the effector-null background of strain PA103, often used to study T3SS effector activity. Inhibition of internalization was associated with T3SS hyperinducibility and ExoS delivery. PA103 fleQ mutation, preventing flagellar assembly, further reduced internalization but did so independently of ExoS. The results revealed intracellular T3SS expression by all strains and suggested that T3SS bistability influences P. aeruginosa internalization. These findings reconcile controversies in the literature surrounding P. aeruginosa internalization and support the principle that P. aeruginosa is not exclusively an extracellular pathogen. Copyright © 2018 Kroken et al.

EMK protein kinase-null mice: dwarfism and hypofertility associated with alterations in the somatotrope and prolactin pathways.

PubMed

Bessone, S; Vidal, F; Le Bouc, Y; Epelbaum, J; Bluet-Pajot, M T; Darmon, M

1999-10-01

Gene trapping was used in embryonic stem (ES) cells in an attempt to inactivate genes involved in development. The Emk (ELKL motif kinase) gene has been disrupted and a mutant mouse line derived. Previous work had shown that EMK kinases, called MARK in the rat, exert a major control on microtubule stability by phosphorylating microtubule-associated proteins and that genes homologous to Emk in yeast or Caenorhabditis elegans are essential for cell and embryonic polarity. Although we found the Emk gene to be active in the preimplantation mouse embryo and then to show a widespread expression, Emk-null mice had no embryonic defect and were viable. They show an overall proportionate dwarfism and a peculiar hypofertility: homozygotes are not fertile when intercrossed, but are fertile in other types of crosses. Insulin-like growth factor I (IGF I) and IGF-binding protein 3 (IGFBP3) were reduced in the plasma of homozygotes of both sexes. A direct implication of the EMK kinase in IGF I plasmatic production is unlikely because the Emk gene does not seem to be expressed in hepatocytes. Nevertheless, GH assayed at arbitrary times in plasma did not show differences between genotypes and GH concentrations in pituitary extracts were not found to be altered in homozygotes. Our results, though, do not exclude the possibility that in the mutants the overall quantity of GH secreted daily is reduced. Our observation of a smaller size of the pituitaries of the mutants is in favor of this hypothesis. The prolactin concentration in the pituitaries was much lowered in homozygous females, but it was normal in males. The possible involvement of EMK protein kinase in hormone secretion in the pituitary and/or the hypothalamus, via the microtubule network, is discussed. Copyright 1999 Academic Press.

Complete or partial reduction of the Met receptor tyrosine kinase in distinct circuits differentially impacts mouse behavior.

PubMed

Thompson, Barbara L; Levitt, Pat

2015-01-01

Our laboratory discovered that the gene encoding the receptor tyrosine kinase, MET, contributes to autism risk. Expression of MET is reduced in human postmortem temporal lobe in autism and Rett Syndrome. Subsequent studies revealed a role for MET in human and mouse functional and structural cortical connectivity. To further understand the contribution of Met to brain development and its impact on behavior, we generated two conditional mouse lines in which Met is deleted from select populations of central nervous system neurons. Mice were then tested to determine the consequences of disrupting Met expression. Mating of Emx1 (cre) and Met (fx/fx) mice eliminates receptor signaling from all cells arising from the dorsal pallium. Met (fx/fx) and Nestin (cre) crosses result in receptor signaling elimination from all neural cells. Behavioral tests were performed to assess cognitive, emotional, and social impairments that are observed in multiple neurodevelopmental disorders and that are in part subserved by circuits that express Met. Met (fx/fx) /Emx1 (cre) null mice displayed significant hypoactivity in the activity chamber and in the T-maze despite superior performance on the rotarod. Additionally, these animals showed a deficit in spontaneous alternation. Surprisingly, Met (fx/fx; fx/+) /Nestin (cre) null and heterozygous mice exhibited deficits in contextual fear conditioning, and Met (fx/+) /Nestin (cre) heterozygous mice spent less time in the closed arms of the elevated plus maze. These data suggest a complex contribution of Met in the development of circuits mediating social, emotional, and cognitive behavior. The impact of disrupting developmental Met expression is dependent upon circuit-specific deletion patterns and levels of receptor activity.

MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity in Caenorhabditis elegans.

PubMed

Ratliff, Miriam; Hill-Harfe, Katherine L; Gleason, Elizabeth J; Ling, Huiping; Kroft, Tim L; L'Hernault, Steven W

2018-05-01

Covalent attachment of ubiquitin to substrate proteins changes their function or marks them for proteolysis, and the specificity of ubiquitin attachment is mediated by the numerous E3 ligases encoded by animals. Mind Bomb is an essential E3 ligase during Notch pathway signaling in insects and vertebrates. While Caenorhabditis elegans encodes a Mind Bomb homolog ( mib-1 ), it has never been recovered in the extensive Notch suppressor/enhancer screens that have identified numerous pathway components. Here, we show that C. elegans mib-1 null mutants have a spermatogenesis-defective phenotype that results in a heterogeneous mixture of arrested spermatocytes, defective spermatids, and motility-impaired spermatozoa. mib-1 mutants also have chromosome segregation defects during meiosis, molecular null mutants are intrinsically temperature-sensitive, and many mib-1 spermatids contain large amounts of tubulin. These phenotypic features are similar to the endogenous RNA intereference (RNAi) mutants, but mib-1 mutants do not affect RNAi. MIB-1 protein is expressed throughout the germ line with peak expression in spermatocytes followed by segregation into the residual body during spermatid formation. C. elegans mib-1 expression, while upregulated during spermatogenesis, also occurs somatically, including in vulva precursor cells. Here, we show that mib-1 mutants suppress both lin-12 and glp-1 ( C. elegans Notch) gain-of-function mutants, restoring anchor cell formation and a functional vulva to the former and partly restoring oocyte production to the latter. However, suppressed hermaphrodites are only observed when grown at 25°, and they are self-sterile. This probably explains why mib-1 was not previously recovered as a Notch pathway component in suppressor/enhancer selection experiments. Copyright © 2018 by the Genetics Society of America.

Fascin 1 is dispensable for developmental and tumour angiogenesis

PubMed Central

Ma, Yafeng; Reynolds, Louise E.; Li, Ang; Stevenson, Richard P.; Hodivala-Dilke, Kairbaan M.; Yamashiro, Shigeko; Machesky, Laura M.

2013-01-01

Summary The actin bundling protein fascin 1 is not expressed in adult epithelial tissues, but during development it is transiently expressed in many different cell types, and later in adults it is expressed in a subset of immune cells, nervous tissues, endothelial cells, smooth muscle cells and pericytes. In contrast to the wealth of knowledge about the role of fascin 1 in cancer cell migration and invasion, little is known about the involvement of fascin 1 in angiogenesis. We speculated that as angiogenesis involves migration and invasion of tissues by endothelial cells, fascin 1 might have a role in both normal and tumour angiogenesis. Here, we provide evidence that loss of fascin 1 causes relatively minor reductions to angiogenesis during embryonic, postnatal and cancerous development by examining E12.5 hindbrains, postnatal retinas and B16F0 tumour cell allografts in fascin 1-null mice. We also find that in fascin 1 null tissues, endothelial cells display reduced filopodia formation during sprouting. We thus propose that fascin 1 expression promotes angiogenesis via filopodia formation, but is largely dispensable for both normal and tumour angiogenesis. PMID:24244855

Fascin 1 is dispensable for developmental and tumour angiogenesis.

PubMed

Ma, Yafeng; Reynolds, Louise E; Li, Ang; Stevenson, Richard P; Hodivala-Dilke, Kairbaan M; Yamashiro, Shigeko; Machesky, Laura M

2013-01-01

The actin bundling protein fascin 1 is not expressed in adult epithelial tissues, but during development it is transiently expressed in many different cell types, and later in adults it is expressed in a subset of immune cells, nervous tissues, endothelial cells, smooth muscle cells and pericytes. In contrast to the wealth of knowledge about the role of fascin 1 in cancer cell migration and invasion, little is known about the involvement of fascin 1 in angiogenesis. We speculated that as angiogenesis involves migration and invasion of tissues by endothelial cells, fascin 1 might have a role in both normal and tumour angiogenesis. Here, we provide evidence that loss of fascin 1 causes relatively minor reductions to angiogenesis during embryonic, postnatal and cancerous development by examining E12.5 hindbrains, postnatal retinas and B16F0 tumour cell allografts in fascin 1-null mice. We also find that in fascin 1 null tissues, endothelial cells display reduced filopodia formation during sprouting. We thus propose that fascin 1 expression promotes angiogenesis via filopodia formation, but is largely dispensable for both normal and tumour angiogenesis.

MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation

PubMed Central

De Luca, Gabriele; Leopardi, Paola; Mancuso, Maria Teresa; Casorelli, Ida; Pichierri, Pietro; Karran, Peter; Bignami, Margherita

2015-01-01

The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients' cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using Mutyh-nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, Mutyh-null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG). Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, Mutyh-null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, Mutyh-null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo. Mutyh−/−mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated Mutyh−/− mice whereas similarly treated wild-type animals remained tumor-free. PMID:25638157

Nrf2 promotes neuronal cell differentiation.

PubMed

Zhao, Fei; Wu, Tongde; Lau, Alexandria; Jiang, Tao; Huang, Zheping; Wang, Xiao-Jun; Chen, Weimin; Wong, Pak Kin; Zhang, Donna D

2009-09-15

The transcription factor Nrf2 has emerged as a master regulator of the endogenous antioxidant response, which is critical in defending cells against environmental insults and in maintaining intracellular redox balance. However, whether Nrf2 has any role in neuronal cell differentiation is largely unknown. In this report, we have examined the effects of Nrf2 on cell differentiation using a neuroblastoma cell line, SH-SY5Y. Retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate, two well-studied inducers of neuronal differentiation, are able to induce Nrf2 and its target gene NAD(P)H quinone oxidoreductase 1 in a dose- and time-dependent manner. RA-induced Nrf2 up-regulation is accompanied by neurite outgrowth and an induction of two neuronal differentiation markers, neurofilament-M and microtubule-associated protein 2. Overexpression of Nrf2 in SH-SY5Y cells promotes neuronal differentiation, whereas inhibition of endogenous Nrf2 expression inhibited neuronal differentiation. More remarkably, the positive role of Nrf2 in neuronal differentiation was verified ex vivo in primary neuron culture. Primary neurons isolated from Nrf2-null mice showed a retarded progress in differentiation, compared to those from wild-type mice. Collectively, our data demonstrate a novel role for Nrf2 in promoting neuronal cell differentiation, which will open new perspectives for therapeutic uses of Nrf2 activators in patients with neurodegenerative diseases.

Analyses of genome wide association data, cytokines, and gene expression in African-Americans with benign ethnic neutropenia

PubMed Central

Shriner, Daniel; Ramos, Edward; Chin, Kyung; Srivastava, Kshitij; Zakai, Neil A.; Cushman, Mary; McClure, Leslie A.; Howard, Virginia; Flegel, Willy A.; Rotimi, Charles N.; Rodgers, Griffin P.

2018-01-01

Background Benign ethnic neutropenia (BEN) is a hematologic condition associated with people of African ancestry and specific Middle Eastern ethnic groups. Prior genetic association studies in large population showed that rs2814778 in Duffy Antigen Receptor for Chemokines (DARC) gene, specifically DARC null red cell phenotype, was associated with BEN. However, the mechanism of this red cell phenotype leading to low white cell count remained elusive. Methods We conducted an extreme phenotype design genome-wide association study (GWAS), analyzed ~16 million single nucleotide polymorphisms (SNP) in 1,178 African-Americans individuals from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study and replicated from 819 African-American participants in the Atherosclerosis Risk in Communities (ARIC) study. Conditional analyses on rs2814778 were performed to identify additional association signals on chromosome 1q22. In a separate cohort of healthy individuals with and without BEN, whole genome gene expression from peripheral blood neutrophils were analyzed for DARC. Results We confirmed that rs2814778 in DARC was associated with BEN (p = 4.09×10−53). Conditioning on rs2814778 abolished other significant chromosome 1 associations. Inflammatory cytokines (IL-2, 6, and 10) in participants in the Howard University Family Study (HUFS) and Multi-Ethnic Study in Atherosclerosis (MESA) showed similar levels in individuals homozygous for the rs2814778 allele compared to others, indicating cytokine sink hypothesis played a minor role in leukocyte homeostasis. Gene expression in neutrophils of individuals with and without BEN was also similar except for low DARC expression in BEN, suggesting normal function. BEN neutrophils had slightly activated profiles in leukocyte migration and hematopoietic stem cell mobilization pathways (expression fold change <2). Conclusions These results in humans support the notion of DARC null erythroid progenitors preferentially differentiating to myeloid cells, leading to activated DARC null neutrophils egressing from circulation to the spleen, and causing relative neutropenia. Collectively, these human data sufficiently explained the mechanism DARC null red cell phenotype causing BEN and further provided a biologic basis that BEN is clinically benign. PMID:29596498

Analyses of genome wide association data, cytokines, and gene expression in African-Americans with benign ethnic neutropenia.

PubMed

Charles, Bashira A; Hsieh, Matthew M; Adeyemo, Adebowale A; Shriner, Daniel; Ramos, Edward; Chin, Kyung; Srivastava, Kshitij; Zakai, Neil A; Cushman, Mary; McClure, Leslie A; Howard, Virginia; Flegel, Willy A; Rotimi, Charles N; Rodgers, Griffin P

2018-01-01

Benign ethnic neutropenia (BEN) is a hematologic condition associated with people of African ancestry and specific Middle Eastern ethnic groups. Prior genetic association studies in large population showed that rs2814778 in Duffy Antigen Receptor for Chemokines (DARC) gene, specifically DARC null red cell phenotype, was associated with BEN. However, the mechanism of this red cell phenotype leading to low white cell count remained elusive. We conducted an extreme phenotype design genome-wide association study (GWAS), analyzed ~16 million single nucleotide polymorphisms (SNP) in 1,178 African-Americans individuals from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study and replicated from 819 African-American participants in the Atherosclerosis Risk in Communities (ARIC) study. Conditional analyses on rs2814778 were performed to identify additional association signals on chromosome 1q22. In a separate cohort of healthy individuals with and without BEN, whole genome gene expression from peripheral blood neutrophils were analyzed for DARC. We confirmed that rs2814778 in DARC was associated with BEN (p = 4.09×10-53). Conditioning on rs2814778 abolished other significant chromosome 1 associations. Inflammatory cytokines (IL-2, 6, and 10) in participants in the Howard University Family Study (HUFS) and Multi-Ethnic Study in Atherosclerosis (MESA) showed similar levels in individuals homozygous for the rs2814778 allele compared to others, indicating cytokine sink hypothesis played a minor role in leukocyte homeostasis. Gene expression in neutrophils of individuals with and without BEN was also similar except for low DARC expression in BEN, suggesting normal function. BEN neutrophils had slightly activated profiles in leukocyte migration and hematopoietic stem cell mobilization pathways (expression fold change <2). These results in humans support the notion of DARC null erythroid progenitors preferentially differentiating to myeloid cells, leading to activated DARC null neutrophils egressing from circulation to the spleen, and causing relative neutropenia. Collectively, these human data sufficiently explained the mechanism DARC null red cell phenotype causing BEN and further provided a biologic basis that BEN is clinically benign.

Rhnull syndrome: identification of a novel mutation in RHce.

PubMed

Rosa, K A; Reid, M E; Lomas-Francis, C; Powell, V I; Costa, F F; Stinghen, S T; Watanabe, A M; Carboni, E K; Baldon, J P; Jucksch, M M F; Castilho, L

2005-11-01

The deficiency of Rh proteins on red blood cells (RBCs) from individuals of the Rh(null) amorph type are the result of homozygosity for a silent RHCE in cis with a deleted RHD. A novel mutation in RHce was identified in two Caucasian Brazilian girls with the amorph type of Rh(null) who were born to parents who were first cousins. RBCs from the Rh(null) sisters and from family members were analyzed by serology and flow cytometry with specific antibodies. Genomic DNA and transcripts were tested by polymerase chain reaction and sequence analysis. Rh(null) RBCs were nonreactive with anti-Rh and anti-LW. Molecular analyses showed a deletion of RHD and of one nucleotide (960/963; GGGG-->GGG) in exon 7 of the RHce. This deletion introduced a frameshift after Gly321, a new C-terminal sequence, and a premature stop codon, resulting in a shorter predicted protein with 357 amino acids. The detection of a unique RHce transcript indicated that the two sisters were homozygous, whereas the other family members were heterozygous for the mutation. A novel mutation resulting in the amorph Rh(null) with loss of Rh antigen expression is described.

Effects of drospirenone-ethinylestradiol and/or metformin on CD4(+)CD28(null) T lymphocytes frequency in women with hyperinsulinemia having polycystic ovary syndrome: a randomized clinical trial.

PubMed

Moro, Francesca; Morciano, Andrea; Tropea, Anna; Sagnella, Francesca; Palla, Carola; Scarinci, Elisa; Ciardulli, Andrea; Martinez, Daniela; Familiari, Alessandra; Liuzzo, Giovanna; Tritarelli, Alessandra; Cosentino, Nicola; Niccoli, Giampaolo; Crea, Filippo; Lanzone, Antonio; Apa, Rosanna

2013-12-01

To evaluate the long-term effects of drospirenone (DRSP)/ethinylestradiol (EE) alone, metformin alone, and DRSP/EE-metformin on CD4(+)CD28(null) T lymphocytes frequency, a cardiovascular risk marker, in patients with hyperinsulinemic polycystic ovary syndrome (PCOS). Randomized clinical trial. Ninety three patients with hyperinsulinemic PCOS were age matched and body mass index matched and randomized to receive a 6 months daily treatment with DRSP (3 mg)/EE (0.03 mg), or metformin (1500 mg), or DRSP/EE combined with metformin. CD4(+)CD28(null) T-cell frequencies. The DRSP/EE and metformin groups did not show any significant change in the CD4(+)CD28(null) frequency compared to the baseline. Interestingly, a statistically significant decrease in CD4(+)CD28(null) frequency occurred after 6 months of DRSP/EE-metformin (median 3-1.5; P < .01). Of note, this statistically significant association was confirmed after adjusting for baseline values in DRSP/EE-metformin group by analysis of covariance (P < .05). In women with hyperinsulinemic PCOS, combined therapy with DRSP/EE and metformin may reduce cardiovascular risk.

A magnesium-dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis.

PubMed

Formstone, Alex; Errington, Jeffery

2005-03-01

MreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB. Construction of an in-frame deletion of mreB and its complementation by mreB(+) only, in trans, established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.

NOSH-Aspirin Inhibits Colon Cancer Cell Growth: Effects Of Positional Isomerism.

PubMed

Vannini, Federica; Kodela, Ravinder; Chattopadhyay, Mitali; Kashfi, Khosrow

2015-08-01

NOSH-aspirin, a novel hybrid that releases nitric oxide (NO) and hydrogen sulfide (H 2 S) was designed to overcome the potential side effects of aspirin. We compared the cell growth inhibitory properties of ortho-, meta-, and para-NOSH-aspirins. Effects of electron donating/withdrawing groups on the stability and biological activity of these novel compounds were also evaluated. Cell line: HT-29 (Cyclooxygenase, COX-1 & -2 expressing) and HCT 15 (COX null) human colon adenocarcimoa; Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G 0 /G 1 ) peak in DNA content histograms; Proliferation: PCNA; ROS: measured hydrogen peroxide and super oxide by flow cytometry using DCFDA and DHE dyes. The IC 50 s for growth inhibition in µM at 24h were, HT-29: ortho-NOSH-ASA (0.04±0.011), meta-NOSH-ASA (0.24±0.11), para-NOSH-ASA (0.46±0.17); significance between the groups were: o vs m P>0.05, o vs p P<0.05, m vs p P>0.05; HCT 15: ortho-NOSH-ASA (0.062±0.006), meta-NOSH-ASA (0.092±0.004), para-NOSH-ASA (0.37±0.04); significance between the groups were: o vs m P<0.01, o vs p P<0.001, m vs p P<0.001. Electron donating/withdrawing groups significantly affected these IC 50 s. All positional isomers qualitatively had similar effects on proliferation, apoptosis, and caused G 0 /G 1 cell cycle arrest in both colon cancer cell lines. The underlying mechanism for these observations appeared to be mediated through ROS, as pretreatment of the cells with N-acetylcysteine, partially blocked these effects. Positional isomerism affects the potency of NOSH-aspirin. The effects appear to be COX independent. Copyright © 2015. Published by Elsevier B.V.

Parathyroid hormone ablation alters erythrocyte parameters that are rescued by calcium-sensing receptor gene deletion

PubMed Central

Romero, Jose R.; Youte, Rodeler; Brown, Edward M.; Pollak, Martin R.; Goltzman, David; Karaplis, Andrew; Pong, Lie-Chin; Chien, Lawrence; Chattopadhyay, Naibedya; Rivera, Alicia

2013-01-01

The mechanisms by which parathyroid hormone (PTH) produces anemia, are unclear. Parathyroid hormone secretion is regulated by the extracellular Ca2+-sensing receptor. We investigated the effects of ablating PTH on hematological indices and erythrocytes volume regulation in wild-type, PTH-null and Ca2+-sensing receptor-null/PTH-null mice. The erythrocyte parameters were measured in whole mouse blood and volume regulatory systems were determined by plasma membrane K+ fluxes and osmotic fragility was measured by hemoglobin determination at varying osmolarities. We observed that the absence of PTH significantly increases mean erythrocyte volume and reticulocyte counts, while decreasing erythrocyte counts, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration. These changes were accompanied by increases in erythrocyte cation content, a denser cell population and increased K+ permeability, which were in part mediated by activation of the K+/Cl− cotransporter and Gardos channel. In addition we observed that erythrocyte osmotic fragility in PTH-null compared with wild-type mice was enhanced. When Ca2+-sensing receptor gene was deleted on the background of PTH-null mice, we observed that several of the alterations in erythrocyte parameters of PTH-null mice were largely rescued, particularly those related to erythrocyte volume, K+ fluxes and osmotic fragility, and became similar to those observed in wild-type mice. Our results demonstrate that Ca2+-sensing receptor and parathyroid hormone are functionally coupled to maintain erythrocyte homeostasis. PMID:23528155

Glutathione S-transferase M1-null genotype as risk factor for SOS in oxaliplatin-treated patients with metastatic colorectal cancer.

PubMed

Vreuls, C P H; Olde Damink, S W M; Koek, G H; Winstanley, A; Wisse, E; Cloots, R H E; van den Broek, M A J; Dejong, C H C; Bosman, F T; Driessen, A

2013-02-19

Oxaliplatin is used as a neo-adjuvant therapy in hepatic colorectal carcinoma metastasis. This treatment has significant side effects, as oxaliplatin is toxic to the sinusoidal endothelial cells and can induce sinusoidal obstruction syndrome (SOS), which is related to decreased overall survival. Glutathione has an important role in the defence system, catalysed by glutathione S-transferase (GST), including two non-enzyme producing polymorphisms (GSTM1-null and GSTT1-null). We hypothesise that patients with a non-enzyme producing polymorphism have a higher risk of developing toxic injury owing to oxaliplatin. In the nontumour-bearing liver, the presence of SOS was studied histopathologically. The genotype was determined by a semi-nested PCR. Thirty-two of the 55 (58%) patients showed SOS lesions, consisting of 27% mild, 22% moderate and 9% severe lesions. The GSTM1-null genotype was present in 25 of the 55 (46%). Multivariate analysis showed that the GSTM1-null genotype significantly correlated with the presence of (moderate-severe) SOS (P=0.026). The GSTM1-null genotype is an independent risk factor for SOS. This finding allows us, in association with other risk factors, to conceive a potential risk profile predicting whether the patient is at risk of developing SOS, before starting oxaliplatin, and subsequently might result in adjustment of treatment.

STAT3 is a negative regulator of granulopoiesis but is not required for G-CSF-dependent differentiation.

PubMed

Lee, Chien-kuo; Raz, Regina; Gimeno, Ramon; Gertner, Rachel; Wistinghausen, Birte; Takeshita, Kenichi; DePinho, Ronald A; Levy, David E

2002-07-01

STAT3 has been described as an essential component of G-CSF-driven cell proliferation and granulopoiesis. This notion was tested by conditional gene ablation in transgenic mice. Contrary to expectation, granulocytes developed from STAT3 null bone marrow progenitors, and STAT3 null neutrophils displayed mature effector functions. Rather than a deficit in granulopoiesis, mice lacking STAT3 in their hematopoietic progenitors developed neutrophilia, and bone marrow cells were hyperresponsive to G-CSF stimulation. These studies provide direct evidence for STAT3-independent granulopoiesis and suggest that STAT3 directs a negative feedback loop necessary for controlling neutrophil numbers, possibly through induced expression of the signaling inhibitor, SOCS3.

Impaired B cell development in the absence of Krüppel-like factor 3.

PubMed

Vu, Thi Thanh; Gatto, Dominique; Turner, Vivian; Funnell, Alister P W; Mak, Ka Sin; Norton, Laura J; Kaplan, Warren; Cowley, Mark J; Agenès, Fabien; Kirberg, Jörg; Brink, Robert; Pearson, Richard C M; Crossley, Merlin

2011-11-15

Krüppel-like factor 3 (Klf3) is a member of the Klf family of transcription factors. Klfs are widely expressed and have diverse roles in development and differentiation. In this study, we examine the function of Klf3 in B cell development by studying B lymphopoiesis in a Klf3 knockout mouse model. We show that B cell differentiation is significantly impaired in the bone marrow, spleen, and peritoneal cavity of Klf3 null mice and confirm that the defects are cell autonomous. In the bone marrow, there is a reduction in immature B cells, whereas recirculating mature cells are noticeably increased. Immunohistology of the spleen reveals a poorly structured marginal zone (MZ) that may in part be caused by deregulation of adhesion molecules on MZ B cells. In the peritoneal cavity, there are significant defects in B1 B cell development. We also report that the loss of Klf3 in MZ B cells is associated with reduced BCR signaling strength and an impaired ability to respond to LPS stimulation. Finally, we show increased expression of a number of Klf genes in Klf3 null B cells, suggesting that a Klf regulatory network may exist in B cells.

Regulation of ecmF gene expression and genetic hierarchy among STATa, CudA, and MybC on several prestalk A-specific gene expressions in Dictyostelium.

PubMed

Saga, Yukika; Inamura, Tomoka; Shimada, Nao; Kawata, Takefumi

2016-05-01

STATa, a Dictyostelium homologue of metazoan signal transducer and activator of transcription, is important for the organizer function in the tip region of the migrating Dictyostelium slug. We previously showed that ecmF gene expression depends on STATa in prestalk A (pstA) cells, where STATa is activated. Deletion and site-directed mutagenesis analysis of the ecmF/lacZ fusion gene in wild-type and STATa null strains identified an imperfect inverted repeat sequence, ACAAATANTATTTGT, as a STATa-responsive element. An upstream sequence element was required for efficient expression in the rear region of pstA zone; an element downstream of the inverted repeat was necessary for sufficient prestalk expression during culmination. Band shift analyses using purified STATa protein detected no sequence-specific binding to those ecmF elements. The only verified upregulated target gene of STATa is cudA gene; CudA directly activates expL7 gene expression in prestalk cells. However, ecmF gene expression was almost unaffected in a cudA null mutant. Several previously reported putative STATa target genes were also expressed in cudA null mutant but were downregulated in STATa null mutant. Moreover, mybC, which encodes another transcription factor, belonged to this category, and ecmF expression was downregulated in a mybC null mutant. These findings demonstrate the existence of a genetic hierarchy for pstA-specific genes, which can be classified into two distinct STATa downstream pathways, CudA dependent and independent. The ecmF expression is indirectly upregulated by STATa in a CudA-independent activation manner but dependent on MybC, whose expression is positively regulated by STATa. © 2016 Japanese Society of Developmental Biologists.

Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes

PubMed Central

Schaetzlein, Sonja; Chahwan, Richard; Avdievich, Elena; Roa, Sergio; Wei, Kaichun; Eoff, Robert L.; Sellers, Rani S.; Clark, Alan B.; Kunkel, Thomas A.; Scharff, Matthew D.; Edelmann, Winfried

2013-01-01

Mammalian Exonuclease 1 (EXO1) is an evolutionarily conserved, multifunctional exonuclease involved in DNA damage repair, replication, immunoglobulin diversity, meiosis, and telomere maintenance. It has been assumed that EXO1 participates in these processes primarily through its exonuclease activity, but recent studies also suggest that EXO1 has a structural function in the assembly of higher-order protein complexes. To dissect the enzymatic and nonenzymatic roles of EXO1 in the different biological processes in vivo, we generated an EXO1-E109K knockin (Exo1EK) mouse expressing a stable exonuclease-deficient protein and, for comparison, a fully EXO1-deficient (Exo1null) mouse. In contrast to Exo1null/null mice, Exo1EK/EK mice retained mismatch repair activity and displayed normal class switch recombination and meiosis. However, both Exo1-mutant lines showed defects in DNA damage response including DNA double-strand break repair (DSBR) through DNA end resection, chromosomal stability, and tumor suppression, indicating that the enzymatic function is required for those processes. On a transformation-related protein 53 (Trp53)-null background, the DSBR defect caused by the E109K mutation altered the tumor spectrum but did not affect the overall survival as compared with p53-Exo1null mice, whose defects in both DSBR and mismatch repair also compromised survival. The separation of these functions demonstrates the differential requirement for the structural function and nuclease activity of mammalian EXO1 in distinct DNA repair processes and tumorigenesis in vivo. PMID:23754438

Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance.

PubMed

Elliott, Michael R; Chekeni, Faraaz B; Trampont, Paul C; Lazarowski, Eduardo R; Kadl, Alexandra; Walk, Scott F; Park, Daeho; Woodson, Robin I; Ostankovich, Marina; Sharma, Poonam; Lysiak, Jeffrey J; Harden, T Kendall; Leitinger, Norbert; Ravichandran, Kodi S

2009-09-10

Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.

The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

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

Yoshino, Tomoko; Maeda, Yasuo; Amagai, Aiko

2007-08-03

Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method.more » Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.« less

Latexin Inactivation Enhances Survival and Long-Term Engraftment of Hematopoietic Stem Cells and Expands the Entire Hematopoietic System in Mice.

PubMed

Liu, Yi; Zhang, Cuiping; Li, Zhenyu; Wang, Chi; Jia, Jianhang; Gao, Tianyan; Hildebrandt, Gerhard; Zhou, Daohong; Bondada, Subbarao; Ji, Peng; St Clair, Daret; Liu, Jinze; Zhan, Changguo; Geiger, Hartmut; Wang, Shuxia; Liang, Ying

2017-04-11

Natural genetic diversity offers an important yet largely untapped resource to decipher the molecular mechanisms regulating hematopoietic stem cell (HSC) function. Latexin (Lxn) is a negative stem cell regulatory gene identified on the basis of genetic diversity. By using an Lxn knockout mouse model, we found that Lxn inactivation in vivo led to the physiological expansion of the entire hematopoietic hierarchy. Loss of Lxn enhanced the competitive repopulation capacity and survival of HSCs in a cell-intrinsic manner. Gene profiling of Lxn-null HSCs showed altered expression of genes enriched in cell-matrix and cell-cell interactions. Thrombospondin 1 (Thbs1) was a potential downstream target with a dramatic downregulation in Lxn-null HSCs. Enforced expression of Thbs1 restored the Lxn inactivation-mediated HSC phenotypes. This study reveals that Lxn plays an important role in the maintenance of homeostatic hematopoiesis, and it may lead to development of safe and effective approaches to manipulate HSCs for clinical benefit. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Role of Na:K:2Cl Cotransporter 1 (NKCC1/SLC12A2) in Dental Epithelium during Enamel Formation in Mice

PubMed Central

Jalali, Rozita; Lodder, Johannes C.; Zandieh-Doulabi, Behrouz; Micha, Dimitra; Melvin, James E.; Catalan, Marcelo A.; Mansvelder, Huibert D.; DenBesten, Pamela; Bronckers, Antonius

2017-01-01

Na+:K+:2Cl− cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl− transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl−-dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl− ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication. PMID:29209227

On the origins of part-time radio pulsars

NASA Astrophysics Data System (ADS)

Zhang, Bing; Gil, Janusz; Dyks, Jaroslaw

2007-01-01

Growing evidence suggests that some radio pulsars only act sporadically. These `part-time' pulsars include long-term nulls, quasi-periodic radio flares in PSR B1931+24, as well as the so-called Rotating Radio Transients (RRATs). Based on the assumption that these objects are isolated neutron stars similar to conventional radio pulsars, we discuss two possible interpretations to the phenomenon. The first interpretation suggests that these objects are pulsars slightly below the radio emission `death line', which become occasionally active only when the conditions for pair production and coherent emission are satisfied. The second interpretation invokes a radio emission direction reversal in conventional pulsars, as has been introduced to interpret the peculiar mode changing phenomenon in PSR B1822-09. In this picture, our line of sight misses the main radio emission beam of the pulsar but happens to sweep the emission beam when the radio emission direction is reversed. These part-time pulsars are therefore the other half of `nulling' pulsars. We suggest that X-ray observations may provide clues to differentiate between these two possibilities.

Loss of ATM kinase activity leads to embryonic lethality in mice.

PubMed

Daniel, Jeremy A; Pellegrini, Manuela; Lee, Baeck-Seung; Guo, Zhi; Filsuf, Darius; Belkina, Natalya V; You, Zhongsheng; Paull, Tanya T; Sleckman, Barry P; Feigenbaum, Lionel; Nussenzweig, André

2012-08-06

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

Dictyostelium LvsB has a regulatory role in endosomal vesicle fusion

PubMed Central

Falkenstein, Kristin; De Lozanne, Arturo

2014-01-01

ABSTRACT Defects in human lysosomal-trafficking regulator (Lyst) are associated with the lysosomal disorder Chediak–Higashi syndrome. The absence of Lyst results in the formation of enlarged lysosome-related compartments, but the mechanism for how these compartments arise is not well established. Two opposing models have been proposed to explain Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, whereas the fusion model identifies Lyst as a negative regulator of fusion between lysosomal vesicles. Here, we used assays that can distinguish between defects in vesicle fusion versus fission. We compared the phenotype of Dictyostelium discoideum cells defective in LvsB, the ortholog of Lyst, with that of two known fission defect mutants (μ3- and WASH-null mutants). We found that the temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and the fusion dynamics of LvsB-null cells are distinct from those of both μ3- and WASH-null fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. PMID:25086066

Endogenous Siderophore 2,5-Dihydroxybenzoic Acid Deficiency Promotes Anemia and Splenic Iron Overload in Mice

PubMed Central

Liu, Zhuoming; Ciocea, Alieta

2014-01-01

Eukaryotes produce a siderophore-like molecule via a remarkably conserved biosynthetic pathway. 3-OH butyrate dehydrogenase (BDH2), a member of the short-chain dehydrogenase (SDR) family of reductases, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore 2,5-dihydroxybenzoic acid (2,5-DHBA). Depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of intracellular iron and mitochondrial iron deficiency in cultured mammalian cells, as well as in yeast cells and zebrafish embryos We disrupted murine bdh2 by homologous recombination to analyze the effect of bdh2 deletion on erythropoiesis and iron metabolism. bdh2 null mice developed microcytic anemia and tissue iron overload, especially in the spleen. Exogenous supplementation with 2,5-DHBA alleviates splenic iron overload in bdh2 null mice. Additionally, bdh2 null mice exhibit reduced serum iron. Although BDH2 has been proposed to oxidize ketone bodies, we found that BDH2 deficiency did not alter ketone body metabolism in vivo. In sum, our findings demonstrate a key role for BDH2 in erythropoiesis. PMID:24777603

Null cosmological singularities and free strings

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

Narayan, K.

2010-03-15

We continue exploring free strings in the background of null Kasner-like cosmological singularities, following K. Narayan, arXiv:0904.4532. We study the free string Schrodinger wave functional along the lines of K. Narayan, arXiv:0807.1517. We find the wave functional to be nonsingular in the vicinity of singularities whose Kasner exponents satisfy certain relations. We compare this with the description in other variables. We then study certain regulated versions of these singularities where the singular region is replaced by a substringy but nonsingular region and study the string spectra in these backgrounds. The string modes can again be solved for exactly, giving somemore » insight into how string oscillator states get excited near the singularity.« less

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

Young, Derek P.; Jacklin, Neil; Punnoose, Ratish J.

Time-reversal is a wave focusing technique that makes use of the reciprocity of wireless propagation channels. It works particularly well in a cluttered environment with associated multipath reflection. This technique uses the multipath in the environment to increase focusing ability. Time-reversal can also be used to null signals, either to reduce unintentional interference or to prevent eavesdropping. It does not require controlled geometric placement of the transmit antennas. Unlike existing techniques it can work without line-of-sight. We have explored the performance of time-reversal focusing in a variety of simulated environments. We have also developed new algorithms to simultaneously focus atmore » a location while nulling at an eavesdropper location. We have experimentally verified these techniques in a realistic cluttered environment.« less

ABCC4 Is a Determinant of Cytarabine-Induced Cytotoxicity and Myelosuppression.

PubMed

Drenberg, C D; Hu, S; Li, L; Buelow, D R; Orwick, S J; Gibson, A A; Schuetz, J D; Sparreboom, A; Baker, S D

2016-02-01

Resistance to cytarabine remains a major challenge in the treatment of acute myeloid leukemia (AML). Based on previous studies implicating ABCC4/MRP4 in the transport of nucleosides, we hypothesized that cytarabine is sensitive to ABCC4-mediated efflux, thereby decreasing its cytotoxic response against AML blasts. The uptake of cytarabine and its monophosphate metabolite was found to be facilitated in ABCC4-expressing vesicles and intracellular retention was significantly impaired by overexpression of human ABCC4 or mouse Abcc4 (P < 0.05). ABCC4 was expressed highly in AML primary blasts and cell lines, and cytotoxicity of cytarabine in cells was increased in the presence of the ABCC4 inhibitors MK571 or sorafenib, as well as after ABCC4 siRNA. In Abcc4-null mice, cytarabine-induced hematological toxicity was enhanced and ex vivo colony-forming assays showed that Abcc4-deficiency sensitized myeloid progenitors to cytarabine. Collectively, these studies demonstrate that ABCC4 plays a protective role against cytarabine-mediated insults in leukemic and host myeloid cells. © 2016 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

The L1-type cell adhesion molecule neuroglian influences the stability of neural ankyrin in the Drosophila embryo but not its axonal localization.

PubMed

Bouley, M; Tian, M Z; Paisley, K; Shen, Y C; Malhotra, J D; Hortsch, M

2000-06-15

Ankyrins are linker proteins, which connect various membrane proteins, including members of the L1 family of neural cell adhesion molecules, with the submembranous actin-spectrin skeleton. Here we report the cloning and characterization of a second, novel Drosophila ankyrin gene (Dank2) that appears to be the result of a gene duplication event during arthropod evolution. The Drosophila L1-type protein neuroglian interacts with products from both Drosophila ankyrin genes. Whereas the previously described ankyrin gene is ubiquitously expressed during embryogenesis, the expression of Dank2 is restricted to the nervous system in the Drosophila embryo. The absence of neuroglian protein in a neuroglian null mutant line causes decreased levels of Dank2 protein in most neuronal cells. This suggests that neuroglian is important for the stability of Dank2 protein. However, neuroglian is not required for Dank2 axonal localization. In temperature-sensitive neuroglian mutants in which neuroglian protein is mislocated at the restrictive temperature to an intracellular location in the neuronal soma, Dank2 protein can still be detected along embryonic nerve tracts.

Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion

PubMed Central

Leoni, Giovanna; Patel, Hetal B.; Sampaio, André L. F.; Gavins, Felicity N. E.; Murray, Joanne F.; Grieco, Paolo; Getting, Stephen J.; Perretti, Mauro

2008-01-01

The existence of anti-inflammatory circuits centered on melanocortin receptors (MCRs) has been supported by the inhibitory properties displayed by melanocortin peptides in models of inflammation and tissue injury. Here we addressed the pathophysiological effect that one MCR, MCR type 3 (MC3R), might have on vascular inflammation. After occlusion (35 min) and reopening of the superior mesenteric artery, MC3R-null mice displayed a higher degree of plasma extravasation (45 min postreperfusion) and cell adhesion and emigration (90 min postreperfusion). These cellular alterations were complemented by higher expression of mesenteric tissue CCL2 and CXCL1 (mRNA and protein) and myeloperoxydase, as compared with wild-type animals. MC1R and MC3R mRNA and protein were both expressed in the inflamed mesenteric tissue; however, no changes in vascular responses were observed in a mouse colony bearing an inactive MC1R. Pharmacological treatment of animals with a selective MC3R agonist ([d-Trp8]-γ-melanocyte-stimulating hormone; 10 μg i.v.) produced marked attenuation of cell adhesion, emigration, and chemokine generation; such effects were absent in MC3R-null mice. These new data reveal the existence of a tonic inhibitory signal provided by MC3R in the mesenteric microcirculation of the mouse, acting to down-regulate cell trafficking and local mediator generation.—Leoni, G., Patel, H. B., Sampaio, A. L. F., Gavins, F. N. E., Murray, J. F., Grieco, P., Getting, S. J., Perretti, M. Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion. PMID:18757499

BmpR1A is a major type 1 BMP receptor for BMP-Smad signaling during skull development.

PubMed

Pan, Haichun; Zhang, Honghao; Abraham, Ponnu; Komatsu, Yoshihiro; Lyons, Karen; Kaartinen, Vesa; Mishina, Yuji

2017-09-01

Craniosynostosis is caused by premature fusion of one or more sutures in an infant skull, resulting in abnormal facial features. The molecular and cellular mechanisms by which genetic mutations cause craniosynostosis are incompletely characterized, and many of the causative genes for diverse types of syndromic craniosynostosis have not yet been identified. We previously demonstrated that augmentation of BMP signaling mediated by a constitutively active BMP type IA receptor (ca-BmpR1A) in neural crest cells (ca1A hereafter) causes craniosynostosis and superimposition of heterozygous null mutation of Bmpr1a rescues premature suture fusion (ca1A;1aH hereafter). In this study, we superimposed heterozygous null mutations of the other two BMP type I receptors, Bmpr1b and Acvr1 (ca1A;1bH and ca1A;AcH respectively hereafter) to further dissect involvement of BMP-Smad signaling. Unlike caA1;1aH, ca1A;1bH and ca1A;AcH did not restore the craniosynostosis phenotypes. In our in vivo study, Smad-dependent BMP signaling was decreased to normal levels in mut;1aH mice. However, BMP receptor-regulated Smads (R-Smads; pSmad1/5/9 hereafter) levels were comparable between ca1A, ca1A;1bH and ca1A;AcH mice, and elevated compared to control mice. Bmpr1a, Bmpr1b and Acvr1 null cells were used to examine potential mechanisms underlying the differences in ability of heterozygosity for Bmpr1a vs. Bmpr1b or Acvr1 to rescue the mut phenotype. pSmad1/5/9 level was undetectable in Bmpr1a homozygous null cells while pSmad1/5/9 levels did not decrease in Bmpr1b or Acvr1 homozygous null cells. Taken together, our study indicates that different levels of expression and subsequent activation of Smad signaling differentially contribute each BMP type I receptor to BMP-Smad signaling and craniofacial development. These results also suggest differential involvement of each type 1 receptor in pathogenesis of syndromic craniosynostoses. Copyright © 2017 Elsevier Inc. All rights reserved.

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

PubMed

Montoya-Durango, Diego E; Ramos, Kenneth A; Bojang, Pasano; Ruiz, Lorell; Ramos, Irma N; Ramos, Kenneth S

2016-01-25

Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

PubMed

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Isocitrate dehydrogenase 1 mutations prime the all-trans retinoic acid myeloid differentiation pathway in acute myeloid leukemia

PubMed Central

Boutzen, Héléna; Saland, Estelle; Larrue, Clément; de Toni, Fabienne; Gales, Lara; Castelli, Florence A.; Cathebas, Mathilde; Zaghdoudi, Sonia; Stuani, Lucille; Kaoma, Tony; Riscal, Romain; Yang, Guangli; Hirsch, Pierre; David, Marion; De Mas-Mansat, Véronique; Delabesse, Eric; Vallar, Laurent; Delhommeau, François; Jouanin, Isabelle; Ouerfelli, Ouathek; Le Cam, Laurent; Linares, Laetitia K.; Junot, Christophe; Portais, Jean-Charles; Vergez, François; Récher, Christian

2016-01-01

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD–Scid–IL2rγnull mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies. PMID:26951332

The dependence of divertor power sharing on magnetic flux balance in near double-null configurations on Alcator C-Mod

NASA Astrophysics Data System (ADS)

Brunner, D.; Kuang, A. Q.; LaBombard, B.; Terry, J. L.

2018-07-01

Management of power exhaust will be a crucial task for tokamak fusion reactors. Reactor concepts are often proposed with double-null divertors, i.e. having two magnetic separatrices in an up-down symmetric configuration. This arrangement is potentially advantageous since the majority of the tokamak exhaust power tends to flow to the outer pair of divertor legs at large major radius, where the geometry is favorable for spreading the heat over a large surface area and there is more room for advanced divertor configurations. Despite the importance, there have been relatively few studies of divertor power sharing in near double null configurations and no studies at the poloidal magnetic fields and scrape-off layer power widths anticipated for a reactor. Motivated by this need we have undertaken a systematic study on Alcator C-Mod, examining the effect of magnetic flux balance on the power sharing among the four divertor legs in near double-null plasmas. Ohmic L-modes at three values of plasma current and ICRF-heated enhanced D-alpha (EDA) H-modes and I-modes at a single value of plasma current are explored, producing poloidal magnetic fields of 0.42, 0.62 and 0.85 Tesla. For Ohmic L-modes and ICRF-heated EDA H-modes, we find that the point of equal power sharing between upper and lower divertors occurs remarkably close to a balanced double null. Power sharing amongst the outer (upper versus lower) and inner (upper versus lower) pairs of divertors can be described in terms of a logistic function of magnetic flux balance, consistent with heat flux mapping along magnetic field lines to the outer midplane. Power sharing between inner and outer legs is found to follow a Gaussian-like function of magnetic flux balance with non-zero power to the inner divertors at double null. The overall behavior of H-modes operated near double null and for I-modes operating to within one heat flux e-folding of double null are found similar to Ohmic L-modes, with a significant reduction of power on the inner divertor legs. The results are encapsulated in terms of empirically-informed analytic functions of magnetic flux balance. When combined with magnetic equilibrium control system specifications, these relationships can be used to specify the power flux handling requirements for each of the four divertor target plates.

The replication defect of ICP0-null mutant herpes simplex virus 1 can be largely complemented by the combined activities of human cytomegalovirus proteins IE1 and pp71.

PubMed

Everett, Roger D; Bell, Adam J; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0.

The Replication Defect of ICP0-Null Mutant Herpes Simplex Virus 1 Can Be Largely Complemented by the Combined Activities of Human Cytomegalovirus Proteins IE1 and pp71

PubMed Central

Bell, Adam J.; Lu, Yongxu; Orr, Anne

2013-01-01

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 is required for efficient lytic infection and productive reactivation from latency and induces derepression of quiescent viral genomes. Despite being unrelated at the sequence level, ICP0 and human cytomegalovirus proteins IE1 and pp71 share some functional similarities in their abilities to counteract antiviral restriction mediated by components of cellular nuclear structures known as ND10. To investigate the extent to which IE1 and pp71 might substitute for ICP0, cell lines were developed that express either IE1 or pp71, or both together, in an inducible manner. We found that pp71 dissociated the hDaxx-ATRX complex and inhibited accumulation of these proteins at sites juxtaposed to HSV-1 genomes but had no effect on the promyelocytic leukemia protein (PML) or Sp100. IE1 caused loss of the small ubiquitin-like modifier (SUMO)-conjugated forms of PML and Sp100 and inhibited the recruitment of these proteins to HSV-1 genome foci but had little effect on hDaxx or ATRX in these assays. Both IE1 and pp71 stimulated ICP0-null mutant plaque formation, but neither to the extent achieved by ICP0. The combination of IE1 and pp71, however, inhibited recruitment of all ND10 proteins to viral genome foci, stimulated ICP0-null mutant HSV-1 plaque formation to near wild-type levels, and efficiently induced derepression of quiescent HSV-1 genomes. These results suggest that ND10-related intrinsic resistance results from the additive effects of several ND10 components and that the effects of IE1 and pp71 on subsets of these components combine to mirror the overall activities of ICP0. PMID:23135716

The Potential for Engineering Enhanced Functional-Feed Soybeans for Sustainable Aquaculture Feed.

PubMed

Herman, Eliot M; Schmidt, Monica A

2016-01-01

Aquaculture is the most rapidly growing segment of global animal production that now surpasses wild-capture fisheries production and is continuing to grow 10% annually. Sustainable aquaculture needs to diminish, and progressively eliminate, its dependence on fishmeal-sourced feed from over-harvested fisheries. Sustainable aquafeed sources will need to be primarily of plant-origin. Soybean is currently the primary global vegetable-origin protein source for aquaculture. Direct exchange of soybean meal for fishmeal in aquafeed has resulted in reduced growth rates due in part to soybean's anti-nutritional proteins. To produce soybeans for use in aquaculture feeds a new conventional line has been bred termed Triple Null by stacking null alleles for the feed-relevant proteins Kunitz Trypsin Inhibitor, lectin, and P34 allergen. Triple Null is now being further enhanced as a platform to build additional transgene traits for vaccines, altered protein composition, and to produce high levels of β-carotene an intrinsic orange-colored aquafeed marker to distinguish the seeds from commodity beans and as the metabolic feedstock precursor of highly valued astaxanthin.

Accentuated Osteoclastic Response to Parathyroid Hormone Undermines Bone Mass Acquisition in Osteonectin-null Mice

PubMed Central

do Reis, Luciene Machado; Kessler, Catherine B.; Adams, Douglas J.; Lorenzo, Joseph; Jorgetti, Vanda; Delany, Anne M.

2008-01-01

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone. In the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 μg/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin-/- mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH. PMID:18499553

The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization

PubMed Central

Lindsey, Stephan; T. Papoutsakis, Eleftherios

2012-01-01

Summary We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. PMID:21226706

Apomorphine, dopamine and phenylethylamine reduce the proportion of phosphorylated insulin receptor substrate 1.

PubMed

Chiarenza, A; Scarselli, M; Novi, F; Lempereur, L; Bernardini, R; Corsini, G U; Maggio, R

2001-12-14

We tested the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the proliferation of fetal calf serum-stimulated human breast cancer (MCF)-7 cells. While the first three compounds were able to block the proliferation of MCF-7 cells, pergolide failed to do so (up to 100 microM). The inhibitory effect of dopamine, apomorphine and phenylethylamine was also evident in serum-starved insulin-stimulated MCF-7 cells. Apomorphine also inhibited the proliferation of the human oestrogen receptor-negative breast cancer (MDA-MB231) and prostate carcinoma (LNCaP) cell lines. In a second set of experiments, we measured the ability of dopamine, apomorphine, phenylethylamine and pergolide to inhibit the phosphorylation (or increase the dephosphorylation) of the insulin receptor substrate (IRS)-1, a major intracellular substrate of the insulin-like growth factor (IGF)-1 receptor. Dopamine, apomorphine and phenylethylamine all reduced to zero the level of phosphorylated IRS-1 with potencies ranging between 0.01 and 1 microM. Finally, we found that fibroblasts from IRS-1 null (-/-) mice were less sensitive to the anti-proliferative effect of apomorphine compared to fibroblasts from wild type-mice, suggesting that the inhibition of IRS-1 phosphorylation by apomorphine is an important aspect of the activity of this compound.

The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization.

PubMed

Lindsey, Stephan; Papoutsakis, Eleftherios T

2011-02-01

We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. © 2011 Blackwell Publishing Ltd.

The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia

PubMed Central

Palomero, Teresa; Dominguez, Maria; Ferrando, Adolfo A.

2008-01-01

Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL. PMID:18414037

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors.

PubMed

Taurino, Marco; Abelenda, Jose A; Río-Alvarez, Isabel; Navarro, Cristina; Vicedo, Begonya; Farmaki, Theodora; Jiménez, Pedro; García-Agustín, Pilar; López-Solanilla, Emilia; Prat, Salomé; Rojo, Enrique; Sánchez-Serrano, José J; Sanmartín, Maite

2014-02-01

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

PubMed Central

Papageorgiou, Angela; Rapley, Joseph; Mesirov, Jill P.; Tamayo, Pablo; Avruch, Joseph

2015-01-01

mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms. PMID:25790369

Binding to Gangliosides Containing N-Acetylneuraminic Acid Is Sufficient To Mediate the Immunomodulatory Properties of the Nontoxic Mucosal Adjuvant LT-IIb(T13I) ▿

PubMed Central

Nawar, Hesham F.; Berenson, Charles S.; Hajishengallis, George; Takematsu, Hiromu; Mandell, Lorrie; Clare, Ragina L.; Connell, Terry D.

2010-01-01

By use of a mouse mucosal immunization model, LT-IIb(T13I), a nontoxic mutant type II heat-labile enterotoxin, was shown to have potent mucosal and systemic adjuvant properties. In contrast to LT-IIb, which binds strongly to ganglioside receptors decorated with either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), LT-IIb(T13I) binds NeuAc gangliosides much less well. Rather, LT-IIb(T13I) binds preferentially to NeuGc gangliosides. To determine if the adjuvant properties of LT-IIb(T13I) are altered in the absence of NeuGc ganglioside receptors, experiments were conducted using a Cmah-null mouse line which is deficient in the synthesis of NeuGc gangliosides. Several immunomodulatory properties of LT-IIb(T13I) were shown to be dependent on NeuGc gangliosides. LT-IIb(T13I) had reduced binding activity for NeuGc-deficient B cells and macrophages; binding to NeuGc-deficient T cells and dendritic cells (DC) was essentially undetectable. Treatment of Cmah-null macrophages with LT-IIb(T13I), however, upregulated the transcription of interleukin-4 (IL-4), IL-6, IL-17, and gamma interferon (IFN-γ), four cytokines important for promoting immune responses. The production of mucosal IgA and serum IgG against an immunizing antigen was augmented in NeuGc-deficient mice administered LT-IIb(T13I) as a mucosal adjuvant. Notably, NeuGc gangliosides are not expressed in humans. Still, treatment of human monocytes with LT-IIb(T13I) induced the secretion of IL-6, an inflammatory cytokine that mediates differential control of leukocyte activation. These results suggested that NeuAc gangliosides are sufficient to mediate the immunomodulatory properties of LT-IIb(T13I) in mice and in human cells. The nontoxic mutant enterotoxin LT-IIb(T13I), therefore, is potentially a new and safe human mucosal adjuvant. PMID:20392887

Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

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

Kimura, Hiroaki; Akiyama, Haruhiko; Nakamura, Takashi

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblasticmore » cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.« less

A DNA segment spanning the mouse Tnfsf11 transcription unit and its upstream regulatory domain rescues the pleiotropic biologic phenotype of the RANKL null mouse.

PubMed

Onal, Melda; Bishop, Kathleen A; St John, Hillary C; Danielson, Allison L; Riley, Erin M; Piemontese, Marilina; Xiong, Jinhu; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

2015-05-01

Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo. © 2014 American Society for Bone and Mineral Research.

Amphiregulin and PTEN evoke a multimodal mechanism of acquired resistance to PI3K inhibition

PubMed Central

Edgar, Kyle A.; Crocker, Lisa; Cheng, Eric; Wagle, Marie-Claire; Wongchenko, Matthew; Yan, Yibing; Wilson, Timothy R.; Dompe, Nicholas; Neve, Richard M.; Belvin, Marcia; Sampath, Deepak; Friedman, Lori S.; Wallin, Jeffrey J.

2014-01-01

Phosphoinositide-3 kinase (PI3K) signaling pathway alterations occur broadly in cancer and PI3K is a promising therapeutic target. Here, we investigated acquired resistance to GDC-0941, a PI3K inhibitor in clinical trials. Colorectal cancer (CRC) cells made to be resistant to GDC-0941 were discovered to secrete amphiregulin, which resulted in increased EGFR/MAPK signaling. Moreover, prolonged PI3K pathway inhibition in cultured cells over a period of months led to a secondary loss of PTEN in 40% of the CRC lines with acquired resistance to PI3K inhibition. In the absence of PI3K inhibitor, these PTEN-null PI3K inhibitor-resistant clones had elevated PI3K pathway signaling and decreased sensitivity to MAPK pathway inhibitors. Importantly, PTEN loss was not able to induce resistance to PI3K inhibitors in the absence of amphiregulin, indicating a multimodal mechanism of acquired resistance. The combination of PI3K and MAPK pathway inhibitors overcame acquired resistance in vitro and in vivo. PMID:25053989

Amphiregulin and PTEN evoke a multimodal mechanism of acquired resistance to PI3K inhibition.

PubMed

Edgar, Kyle A; Crocker, Lisa; Cheng, Eric; Wagle, Marie-Claire; Wongchenko, Matthew; Yan, Yibing; Wilson, Timothy R; Dompe, Nicholas; Neve, Richard M; Belvin, Marcia; Sampath, Deepak; Friedman, Lori S; Wallin, Jeffrey J

2014-03-01

Phosphoinositide-3 kinase (PI3K) signaling pathway alterations occur broadly in cancer and PI3K is a promising therapeutic target. Here, we investigated acquired resistance to GDC-0941, a PI3K inhibitor in clinical trials. Colorectal cancer (CRC) cells made to be resistant to GDC-0941 were discovered to secrete amphiregulin, which resulted in increased EGFR/MAPK signaling. Moreover, prolonged PI3K pathway inhibition in cultured cells over a period of months led to a secondary loss of PTEN in 40% of the CRC lines with acquired resistance to PI3K inhibition. In the absence of PI3K inhibitor, these PTEN-null PI3K inhibitor-resistant clones had elevated PI3K pathway signaling and decreased sensitivity to MAPK pathway inhibitors. Importantly, PTEN loss was not able to induce resistance to PI3K inhibitors in the absence of amphiregulin, indicating a multimodal mechanism of acquired resistance. The combination of PI3K and MAPK pathway inhibitors overcame acquired resistance in vitro and in vivo.

PTEN is a protein phosphatase that targets active PTK6 and inhibits PTK6 oncogenic signaling in prostate cancer.

PubMed

Wozniak, Darren J; Kajdacsy-Balla, Andre; Macias, Virgilia; Ball-Kell, Susan; Zenner, Morgan L; Bie, Wenjun; Tyner, Angela L

2017-11-15

PTEN activity is often lost in prostate cancer. We show that the tyrosine kinase PTK6 (BRK) is a PTEN substrate. Phosphorylation of PTK6 tyrosine 342 (PY342) promotes activation, while phosphorylation of tyrosine 447 (PY447) regulates auto-inhibition. Introduction of PTEN into a PTEN null prostate cancer cell line leads to dephosphorylation of PY342 but not PY447 and PTK6 inhibition. Conversely, PTEN knockdown promotes PTK6 activation in PTEN positive cells. Using a variety of PTEN mutant constructs, we show that protein phosphatase activity of PTEN targets PTK6, with efficiency similar to PTP1B, a phosphatase that directly dephosphorylates PTK6 Y342. Conditional disruption of Pten in the mouse prostate leads to tumorigenesis and increased phosphorylation of PTK6 Y342, and disruption of Ptk6 impairs tumorigenesis. In human prostate tumor tissue microarrays, loss of PTEN correlates with increased PTK6 PY342 and poor outcome. These data suggest PTK6 activation promotes invasive prostate cancer induced by PTEN loss.

Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

PubMed

Mamoon, Abulkhair; Subauste, Angela; Subauste, Maria C; Subauste, Jose

2014-10-25

Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver. Copyright © 2014. Published by Elsevier B.V.

The Relation between Reconnected Flux, the Parallel Electric Field, and the Reconnection Rate in a Three-Dimensional Kinetic Simulation of Magnetic Reconnection

NASA Astrophysics Data System (ADS)

Wendel, D. E.; Olson, D. K.; Hesse, M.; Karimabadi, H.; Daughton, W. S.

2013-12-01

We investigate the distribution of parallel electric fields and their relationship to the location and rate of magnetic reconnection of a large particle-in-cell simulation of 3D turbulent magnetic reconnection with open boundary conditions. The simulation's guide field geometry inhibits the formation of topological features such as separators and null points. Therefore, we derive the location of potential changes in magnetic connectivity by finding the field lines that experience a large relative change between their endpoints, i.e., the quasi-separatrix layer. We find a correspondence between the locus of changes in magnetic connectivity, or the quasi-separatrix layer, and the map of large gradients in the integrated parallel electric field (or quasi-potential). Furthermore, we compare the distribution of parallel electric fields along field lines with the reconnection rate. We find the reconnection rate is controlled by only the low-amplitude, zeroth and first-order trends in the parallel electric field, while the contribution from high amplitude parallel fluctuations, such as electron holes, is negligible. The results impact the determination of reconnection sites within models of 3D turbulent reconnection as well as the inference of reconnection rates from in situ spacecraft measurements. It is difficult through direct observation to isolate the locus of the reconnection parallel electric field amidst the large amplitude fluctuations. However, we demonstrate that a positive slope of the partial sum of the parallel electric field along the field line as a function of field line length indicates where reconnection is occurring along the field line.

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

Nonobese Diabetic (NOD) Mice Congenic for a Targeted Deletion of 12/15-Lipoxygenase Are Protected From Autoimmune Diabetes

PubMed Central

McDuffie, Marcia; Maybee, Nelly A.; Keller, Susanna R.; Stevens, Brian K.; Garmey, James C.; Morris, Margaret A.; Kropf, Elizabeth; Rival, Claudia; Ma, Kaiwen; Carter, Jeffrey D.; Tersey, Sarah A.; Nunemaker, Craig S.; Nadler, Jerry L.

2010-01-01

OBJECTIVE 12/15-lipoxygenase (12/15-LO), one of a family of fatty acid oxidoreductase enzymes, reacts with polyenoic fatty acids to produce proinflammatory lipids. 12/15-LO is expressed in macrophages and pancreatic β-cells. It enhances interleukin 12 production by macrophages, and several of its products induce apoptosis of β-cells at nanomolar concentrations in vitro. We had previously demonstrated a role for 12/15-LO in β-cell damage in the streptozotocin model of diabetes. Since the gene encoding 12/15-LO (gene designation Alox15) lies within the Idd4 diabetes susceptibility interval in NOD mice, we hypothesized that 12/15-LO is also a key regulator of diabetes susceptibility in the NOD mouse. RESEARCH DESIGN AND METHODS We developed NOD mice carrying an inactivated 12/15-LO locus (NOD-Alox15null) using a “speed congenic” protocol, and the mice were monitored for development of insulitis and diabetes. RESULTS NOD mice deficient in 12/15-LO develop diabetes at a markedly reduced rate compared with NOD mice (2.5 vs. >60% in females by 30 weeks). Nondiabetic female NOD-Alox15null mice demonstrate improved glucose tolerance, as well as significantly reduced severity of insulitis and improved β-cell mass, when compared with age-matched nondiabetic NOD females. Disease resistance is associated with decreased numbers of islet-infiltrating activated macrophages at 4 weeks of age in NOD-Alox15null mice, preceding the development of insulitis. Subsequently, islet-associated infiltrates are characterized by decreased numbers of CD4+ T cells and increased Foxp3+ cells. CONCLUSIONS These results suggest an important role for 12/15-LO in conferring susceptibility to autoimmune diabetes in NOD mice through its effects on macrophage recruitment or activation. PMID:17940120

Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.

PubMed

Zagorski, Joseph W; Maser, Tyler P; Liby, Karen T; Rockwell, Cheryl E

2017-05-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert -butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NF κ B activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NF κ B activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NF κ B activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NF κ B and Nrf2. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

NASA Technical Reports Server (NTRS)

Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

2001-01-01

Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

Frequency of null allele of Human Leukocyte Antigen-G (HLA-G) locus in subjects to recurrent miscarriage.

PubMed

Alizadeh, Nazila; Mosaferi, Elnaz; Farzadi, Laya; Majidi, Jafar; Monfaredan, Amir; Yousefi, Bahman; Baradaran, Behzad

2016-07-01

Human leukocyte antigen-G (HLA-G) is a non-classical class I molecule highly expressed by extravillous cytotrophoblast cells. Due to a single base pair deletion, its function can be compensated by other isoforms. Investigating the frequency of null allele in Recurrent Miscarriage (RM) subjects could be useful in understanding the relationship between frequency of this allele and RM in a given population. This study aimed to determine the frequency of HLA-G*0105N null allele and its potential association with down-regulation of HLA-G in subjects with RM. Western blotting was used to assess the level of HLA-G protein expression. For investigating the frequency of HLA-G*0105N null allele in RM subjects, PCR-RFLP method was used. Exon 3 of HLA-G gene was amplified by polymerase chain reaction (PCR). Subsequently, PpuM-1 enzyme was employed to digest the PCR products and fragments were analyzed using gel electrophoresis. Digestion using restriction enzyme showed the presence of heterozygous HLA-G*0105N null allele in 10% of the test population. Western blotting results confirmed the decrease in expression of HLA-G in the placental tissue of subjects with RM compared to subjects who could give normal birth. The frequency of heterozygous HLA-G*0105N null allele was high to some extent in subjects with RM. The mutation rate in subjects suggested that there is a significant association between RM and frequency of mutations in this allele.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos

PubMed Central

Dackor, J.; Strunk, K. E.; Wehmeyer, M. M.; Threadgill, D. W.

2007-01-01

Homozygosity for the Egfrtm1Mag null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta was quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfrtm1Mag homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation. PMID:17822758

Parathyroid hormone ablation alters erythrocyte parameters that are rescued by calcium-sensing receptor gene deletion.

PubMed

Romero, Jose R; Youte, Rodeler; Brown, Edward M; Pollak, Martin R; Goltzman, David; Karaplis, Andrew; Pong, Lie-Chin; Chien, Lawrence; Chattopadhyay, Naibedya; Rivera, Alicia

2013-07-01

The mechanisms by which parathyroid hormone (PTH) produces anemia are unclear. Parathyroid hormone secretion is regulated by the extracellular Ca2+ -sensing receptor. We investigated the effects of ablating PTH on hematological indices and erythrocytes volume regulation in wild-type, PTH-null, and Ca2+ -sensing receptor-null/PTH-null mice. The erythrocyte parameters were measured in whole mouse blood, and volume regulatory systems were determined by plasma membrane K+ fluxes, and osmotic fragility was measured by hemoglobin determination at varying osmolarities. We observed that the absence of PTH significantly increases mean erythrocyte volume and reticulocyte counts, while decreasing erythrocyte counts, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration. These changes were accompanied by increases in erythrocyte cation content, a denser cell population, and increased K+ permeability, which were in part mediated by activation of the K+ /Cl- cotransporter and Gardos channel. In addition we observed that erythrocyte osmotic fragility in PTH-null compared with wild-type mice was enhanced. When Ca2+ -sensing receptor gene was deleted on the background of PTH-null mice, we observed that several of the alterations in erythrocyte parameters of PTH-null mice were largely rescued, particularly those related to erythrocyte volume, K+ fluxes and osmotic fragility, and became similar to those observed in wild-type mice. Our results demonstrate that Ca2+ -sensing receptor and parathyroid hormone are functionally coupled to maintain erythrocyte homeostasis. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The plasminogen activator system modulates sympathetic nerve function.

PubMed

Schaefer, Ulrich; Machida, Takuji; Vorlova, Sandra; Strickland, Sidney; Levi, Roberto

2006-09-04

Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA-null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P<0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)-null mice released much more NE (i.e., a 275% increase; P<0.05). Furthermore, vasa deferentia from t-PA-null mice were hyporesponsive to EFS (P<0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1-null mice were much more responsive (P<0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA-null than in WT control mice (P<0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P<0.05) in t-PA-null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.

ESA to test the smartest technique for detecting extrasolar planets from the ground

NASA Astrophysics Data System (ADS)

2002-03-01

GENIE will use ESO's Very Large Telescopes Credits: European Southern Observatory This photo shows an aerial view of the observing platform on the top of Paranal mountain (from late 1999), with the four enclosu Three 1.8-m VLTI Auxiliary Telescopes (ATs) and paths of the light beams have been superposed on the photo. Also seen are some of the 30 'stations' where the ATs will be positioned for observations and from where the light beams from the telescopes can enter the Interferometric Tunnel below. The straight structures are supports for the rails on which the telescopes can move from one station to another. The Interferometric Laboratory (partly subterranean) is at the centre of the platform. How nulling interferometry works Credits: ESA 2002/Medialab How nulling interferometry works In nulling interferometry, light from a distant star (red beams) hits each telescope, labelled T1 and T2, simultaneously. Before the resultant light beams are combined, the beam from one telescope is delayed by half a wavelength. This means that when the rays are brought together, peaks from one telescope line up with troughs from the other and so are cancelled out (represented by the straight red line), leaving no starlight. Light from a planet (blue beams), orbiting the star, enters the telescopes at an angle. This introduces a delay in the light reaching the second telescope. So, even after the half wavelength change in one of the rays, when the beams are combined they are reinforced (represented by the large blue waves) rather than cancelled out. Illustration by Medialab. Nulling interferometry combines the signal from a number of different telescopes in such a way that the light from the central star is cancelled out, leaving the much fainter planet easier to see. This is possible because light is a wave with peaks and troughs. Usually when combining light from two or more telescopes, a technique called interferometry, the peaks are lined up with one another to boost the signal. In nulling interferometry, however, the peaks are lined up with the troughs so they cancel out to nothing and the star disappears. Planets in orbit around the star show up, however, because they are offset from the central star and their light takes different paths through the telescope system. ESA and ESO will build a new instrument called GENIE (Ground-based European Nulling Interferometer Experiment) to perform nulling interferometry using ESO's Very Large Telescope (VLT), a collection of four 8-metre telescopes in Chile. It will be the biggest investigation of nulling interferometry to date. "It's being tested in the lab in a number of places but we can do more," says Malcolm Fridlund, project scientist for the Darwin mission at the European Space Research and Technology Centre, the Netherlands. "We intend to use the world's largest telescope and the world's largest interferometer to get very high resolution." Using GENIE to perfect this technique will provide invaluable information for engineers about how to build the 'hub' spacecraft of the Darwin flotilla. Scheduled for launch in the middle of the next decade Darwin is a collection of six space telescopes and two other spacecraft, which will together search for Earth-like planets around nearby stars. The hub will combine the light from the telescopes. "If you see the way of getting to Darwin as being outlined by a number of technological milestones this is one of the most important ones," says Malcolm Fridlund. Once up and running, GENIE will also provide a training ground for astronomers who will later use Darwin. For example, it will allow them to perfect their methods of interpreting Darwin data because, as well as the engineering tests, GENIE will be capable of real science. One of its greatest tasks will be to develop the target list of stars for Darwin to study. As recently discovered by ESA's Ulysses spaceprobe, the signature of a planetary system is probably a ring of dust surrounding the central star. GENIE will be able to look for these dust rings and make sure that the dust is not so dense that it will mask the planets from view. GENIE will see failed stars, known as brown dwarfs and, if the instrument performs to expectations, may also see some of the already-discovered giant planets. So far, these worlds have never been seen, only inferred to exist by the effect they have on their parent stars. From Earth, two things handicap nulling interferometry. Firstly, the atmosphere smears out the starlight so that its cancellation is a hundred times less effective than it will be in space. Secondly, planets are most easily seen using infrared wavelengths because they are warm. So, observing from the surface of Earth, itself a planet emitting infrared radiation, is like peering through fog. In space, these two problems disappear and Darwin will be able to see smaller, Earth-like worlds. "We have calculated that with Darwin we could see an 'Earth' if it were ten light-years away with a few hours of observation time. With the VLT, it would be impossible because of the atmosphere. Even if the atmosphere weren't there it would take 450 days because of the infrared background released by the Earth. So we have to go into space," says Fridlund. GENIE is expected to be on-line by 2006.

Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

1997-01-01

A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

No dramatic age-related loss of hair cells and spiral ganglion neurons in Bcl-2 over-expression mice or Bax null mice

PubMed Central

2010-01-01

Age-related decline of neuronal function is associated with age-related structural changes. In the central nervous system, age-related decline of cognitive performance is thought to be caused by synaptic loss instead of neuronal loss. However, in the cochlea, age-related loss of hair cells and spiral ganglion neurons (SGNs) is consistently observed in a variety of species, including humans. Since age-related loss of these cells is a major contributing factor to presbycusis, it is important to study possible molecular mechanisms underlying this age-related cell death. Previous studies suggested that apoptotic pathways were involved in age-related loss of hair cells and SGNs. In the present study, we examined the role of Bcl-2 gene in age-related hearing loss. In one transgenic mouse line over-expressing human Bcl-2, there were no significant differences between transgenic mice and wild type littermate controls in their hearing thresholds during aging. Histological analysis of the hair cells and SGNs showed no significant conservation of these cells in transgenic animals compared to the wild type controls during aging. These data suggest that Bcl-2 overexpression has no significant effect on age-related loss of hair cells and SGNs. We also found no delay of age-related hearing loss in mice lacking Bax gene. These findings suggest that age-related hearing loss is not through an apoptotic pathway involving key members of Bcl-2 family. PMID:20637089

Defective Autophagosome Formation in p53-Null Colorectal Cancer Reinforces Crocin-Induced Apoptosis

PubMed Central

Amin, Amr; Bajbouj, Khuloud; Koch, Adrian; Gandesiri, Muktheshwar; Schneider-Stock, Regine

2015-01-01

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53−/− after 24 h. Crocin induced inefficient autophagy in HCT116 p53−/− cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53−/− after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53−/− cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death. PMID:25584615

Changes in p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.

PubMed

Alexandrova, A; Ivanov, A; Chumakov, P; Kopnin, B; Vasiliev, J

2000-11-23

Effects of p53 expression on cell morphology and motility were studied using the derivatives of p53-null 10(1) mouse fibroblasts with tetracycline-regulated expression of exogenous human p53. Induction of p53 expression was accompanied by significant decrease in extracellular matrix (fibronectin) and reduction of matrix fibrils, diminution of the number and size of focal contacts, decrease of cell areas, establishment of more elongated cell shape and alterations of actin cytoskeleton (actin bundles became thinner, their number and size decreased). Expression of His175 and Gln22/ Ser23 p53 mutants caused no such effects. To study the influence of p53 expression on cell motility we used wound technique and videomicroscopy observation of single living cells. It was found that induction of p53 expression led to increase of lamellar activity of cell edge. However, in spite of enhanced lamellar activity p53-expressing cells migrated to shorter distance and filled the narrow wound in longer time as compared with their p53-null counterparts. Possible mechanisms of the influence of p53 expression on cell morphology and motility are discussed.

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder.

PubMed

Torino, J L; Burger, M S; Reznikoff, C A; Swaminathan, S

2001-01-01

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.