A Disruption of ctpA Encoding Carboxy-Terminal Protease Attenuates Burkholderia mallei and Induces Partial Protection in CD1 Mice

DTIC Science & Technology

2008-06-03

etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular...disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in...IgG2a and were partially protected against chal- lenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

PubMed

Katayama, Takahiro; Yasukawa, Hiro

2008-10-01

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

PubMed

Gangaiah, Dharanesh; Zhang, Xinjun; Baker, Beth; Fortney, Kate R; Gao, Hongyu; Holley, Concerta L; Munson, Robert S; Liu, Yunlong; Spinola, Stanley M

2016-05-01

Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

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

Klein, George, E-mail: Georg.Klein@ki.se; Klein, Eva; Kashuba, Elena

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore asmore » a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.« less

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

PubMed

Echenique-Rivera, Hebert; Muzzi, Alessandro; Del Tordello, Elena; Seib, Kate L; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-05-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

PubMed Central

Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-01-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism. PMID:21589640

Cell transformation by human adenoviruses.

PubMed

Endter, C; Dobner, T

2004-01-01

The last 40 years of molecular biological investigations into human adenoviruses have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of their productive infection cycle in permissive host cells. Also, initial observations concerning the carcinogenic potential of human adenoviruses subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer, and established adenoviruses as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human adenoviruses is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in adenovirus-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, detailed studies on the tumorigenic potential of subgroup D adenovirus type 9 (Ad9) E4 have now revealed a new pathway that points to a novel, general mechanism of virus-mediated oncogenesis. In this chapter, we summarize the current state of knowledge about the oncogenes and oncogene products of human adenoviruses, focusing particularly on recent findings concerning the transforming and oncogenic properties of viral proteins encoded in the E1B and E4 transcription units.

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium. PMID:21572528

RNA-Seq analysis of isolate- and growth phase-specific differences in the global transcriptomes of enteropathogenic Escherichia coli prototype isolates

PubMed Central

Hazen, Tracy H.; Daugherty, Sean C.; Shetty, Amol; Mahurkar, Anup A.; White, Owen; Kaper, James B.; Rasko, David A.

2015-01-01

Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrheal illness among infants in developing countries. E. coli isolates classified as typical EPEC are identified by the presence of the locus of enterocyte effacement (LEE) and the bundle-forming pilus (BFP), and absence of the Shiga-toxin genes, while the atypical EPEC also encode LEE but do not encode BFP or Shiga-toxin. Comparative genomic analyses have demonstrated that EPEC isolates belong to diverse evolutionary lineages and possess lineage- and isolate-specific genomic content. To investigate whether this genomic diversity results in significant differences in global gene expression, we used an RNA sequencing (RNA-Seq) approach to characterize the global transcriptomes of the prototype typical EPEC isolates E2348/69, B171, C581-05, and the prototype atypical EPEC isolate E110019. The global transcriptomes were characterized during laboratory growth in two different media and three different growth phases, as well as during adherence of the EPEC isolates to human cells using in vitro tissue culture assays. Comparison of the global transcriptomes during these conditions was used to identify isolate- and growth phase-specific differences in EPEC gene expression. These analyses resulted in the identification of genes that encode proteins involved in survival and metabolism that were coordinately expressed with virulence factors. These findings demonstrate there are isolate- and growth phase-specific differences in the global transcriptomes of EPEC prototype isolates, and highlight the utility of comparative transcriptomics for identifying additional factors that are directly or indirectly involved in EPEC pathogenesis. PMID:26124752

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

PubMed

Lemaître, Chloé; Bidet, Philippe; Bingen, Edouard; Bonacorsi, Stéphane

2012-06-21

The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation

PubMed Central

1994-01-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold- sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase- encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth. PMID:7962097

Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation.

PubMed

Kim, Y J; Francisco, L; Chen, G C; Marcotte, E; Chan, C S

1994-12-01

The IPL2 gene is known to be required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. We now show that IPL2 is identical to the previously identified BEM2 gene. bem2 mutants are defective in bud site selection at 26 degrees C and localized cell surface growth and organization of the actin cytoskeleton at 37 degrees C. BEM2 encodes a protein with a COOH-terminal domain homologous to sequences found in several GTPase-activating proteins, including human Bcr. The GTPase-activating protein-domain from the Bem2 protein (Bem2p) or human Bcr can functionally substitute for Bem2p. The Rho1 and Rho2 GTPases are the likely in vivo targets of Bem2p because bem2 mutant phenotypes can be partially suppressed by increasing the gene dosage of RHO1 or RHO2. CDC55 encodes the putative regulatory B subunit of protein phosphatase 2A, and mutations in BEM2 have previously been identified as suppressors of the cdc55-1 mutation. We show here that mutations in the previously identified GRR1 gene can suppress bem2 mutations. grr1 and cdc55 mutants are both elongated in shape and cold-sensitive for growth, and cells lacking both GRR1 and CDC55 exhibit a synthetic lethal phenotype. bem2 mutant phenotypes also can be suppressed by the SSD1-vl (also known as SRK1) mutation, which was shown previously to suppress mutations in the protein phosphatase-encoding SIT4 gene. Cells lacking both BEM2 and SIT4 exhibit a synthetic lethal phenotype even in the presence of the SSD1-v1 suppressor. These genetic interactions together suggest that protein phosphorylation and dephosphorylation play an important role in the BEM2-mediated process of polarized cell growth.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

AAV vector encoding human VEGF165-transduced pectineus muscular flaps increase the formation of new tissue through induction of angiogenesis in an in vivo chamber for tissue engineering: A technique to enhance tissue and vessels in microsurgically engineered tissue.

PubMed

Moimas, Silvia; Manasseri, Benedetto; Cuccia, Giuseppe; Stagno d'Alcontres, Francesco; Geuna, Stefano; Pattarini, Lucia; Zentilin, Lorena; Giacca, Mauro; Colonna, Michele R

2015-01-01

In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen-glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine.

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Expression and rearrangement of the ROS1 gene in human glioblastoma cells

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

Birchmeier, C.; Sharma, S.; Wigler, M.

1987-12-01

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, the authors found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, they detected a potentially activating mutation at the ROS1 locus.

Tumor-Protective Mechanism Identified from Premature Aging Disease | Center for Cancer Research

Cancer.gov

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extraordinarily rare genetic disorder caused by a mutation in the LMNA gene, which encodes architectural proteins of the human cell nucleus. The mutation causes the production of a mutant protein called progerin. Patients with HGPS display signs of premature aging, such as hair loss, slowed growth, weakening of bone and joint

Human Hrs, a tyrosine kinase substrate in growth factor-stimulated cells: cDNA cloning and mapping of the gene to chromosome 17.

PubMed

Lu, L; Komada, M; Kitamura, N

1998-06-15

Hrs is a 115kDa zinc finger protein which is rapidly tyrosine phosphorylated in cells stimulated with various growth factors. We previously purified the protein from a mouse cell line and cloned its cDNA. In the present study, we cloned a human Hrs cDNA from a human placenta cDNA library by cross-hybridization, using the mouse cDNA as a probe, and determined its nucleotide sequence. The human Hrs cDNA encoded a 777-amino-acid protein whose sequence was 93% identical to that of mouse Hrs. Northern blot analysis showed that the Hrs mRNA was about 3.0kb long and was expressed in all the human adult and fetal tissues tested. In addition, we showed by genomic Southern blot analysis that the human Hrs gene was a single-copy gene with a size of about 20kb. Furthermore, the human Hrs gene was mapped to chromosome 17 by Southern blotting of genomic DNAs from human/rodent somatic cell hybrids. Copyright 1998 Elsevier Science B.V. All rights reserved.

A pituitary gene encodes a protein that produces differentiation of breast and prostate cancer cells.

PubMed

Platica, Micsunica; Ivan, Elena; Holland, James F; Ionescu, Alin; Chen, Sheryl; Mandeli, John; Unger, Pamela D; Platica, Ovidiu

2004-02-10

A cDNA clone of 1.1 kb encoding a 108-aa polypeptide was isolated from a human pituitary cDNA library by expression cloning. This protein was named tumor differentiation factor (TDF). The recombinant TDF protein and a 20-aa peptide, P1, selected from the ORF of the gene, induced morphological and biochemical changes consistent with differentiation of human breast and prostate cancer cells. Fibroblast, kidney, hepatoma, and leukemic lymphocytic cell lines were unaffected. Breast and prostate cancer cells aggregated in spheroid-like structures within 24 h of exposure to TDF. This effect was abrogated by a specific affinity-purified rabbit polyclonal anti-P1 Ab. E-cadherin expression was increased in a dose-dependent manner by TDF. Treatment of MCF7 cells with TDF led to production of a lactalbumin-related protein. Peptide P1 significantly decreased the growth of androgen-independent DU145 prostate cancer in severe combined immunodeficient mice. The presence of TDF protein in human sera was detected by the anti-P1 Ab, suggesting a role of TDF in endocrine metabolism. The fact that all activities of TDF can be mimicked by a peptide derived from the encoding TDF sequence opens the possibility of therapeutic applications.

A Molecular Basis for Bifidobacterial Enrichment in the Infant Gastrointestinal Tract123

PubMed Central

Garrido, Daniel; Barile, Daniela; Mills, David A.

2012-01-01

Bifidobacteria are commonly used as probiotics in dairy foods. Select bifidobacterial species are also early colonizers of the breast-fed infant colon; however, the mechanism for this enrichment is unclear. We previously showed that Bifidobacterium longum subsp. infantis is a prototypical bifidobacterial species that can readily utilize human milk oligosaccharides as the sole carbon source. MS-based glycoprofiling has revealed that numerous B. infantis strains preferentially consume small mass oligosaccharides, abundant in human milks. Genome sequencing revealed that B. infantis possesses a bias toward genes required to use mammalian-derived carbohydrates. Many of these genomic features encode enzymes that are active on milk oligosaccharides including a novel 40-kb region dedicated to oligosaccharide utilization. Biochemical and molecular characterization of the encoded glycosidases and transport proteins has further resolved the mechanism by which B. infantis selectively imports and catabolizes milk oligosaccharides. Expression studies indicate that many of these key functions are only induced during growth on milk oligosaccharides and not expressed during growth on other prebiotics. Analysis of numerous B. infantis isolates has confirmed that these genomic features are common among the B. infantis subspecies and likely constitute a competitive colonization strategy used by these unique bifidobacteria. By detailed characterization of the molecular mechanisms responsible, these studies provide a conceptual framework for bifidobacterial persistence and host interaction in the infant gastrointestinal tract mediated in part through consumption of human milk oligosaccharides. PMID:22585920

Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids

PubMed Central

Hoedt, Emily C; Cuív, Páraic Ó; Evans, Paul N; Smith, Wendy J M; McSweeney, Chris S; Denman, Stuart E; Morrison, Mark

2016-01-01

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091T, strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091T genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091T genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these ‘low-methane-emitting' herbivores. PMID:27022996

Differences down-under: alcohol-fueled methanogenesis by archaea present in Australian macropodids.

PubMed

Hoedt, Emily C; Cuív, Páraic Ó; Evans, Paul N; Smith, Wendy J M; McSweeney, Chris S; Denman, Stuart E; Morrison, Mark

2016-10-01

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091(T), strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091(T) genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091(T) genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these 'low-methane-emitting' herbivores.

Growth control of the eukaryote cell: a systems biology study in yeast.

PubMed

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David Cj; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom Pj; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.

Growth control of the eukaryote cell: a systems biology study in yeast

PubMed Central

Castrillo, Juan I; Zeef, Leo A; Hoyle, David C; Zhang, Nianshu; Hayes, Andrew; Gardner, David CJ; Cornell, Michael J; Petty, June; Hakes, Luke; Wardleworth, Leanne; Rash, Bharat; Brown, Marie; Dunn, Warwick B; Broadhurst, David; O'Donoghue, Kerry; Hester, Svenja S; Dunkley, Tom PJ; Hart, Sarah R; Swainston, Neil; Li, Peter; Gaskell, Simon J; Paton, Norman W; Lilley, Kathryn S; Kell, Douglas B; Oliver, Stephen G

2007-01-01

Background Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. Results Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. Conclusion This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell. PMID:17439666

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae

PubMed Central

Ipe, Deepak S.; Ben Zakour, Nouri L.; Sullivan, Matthew J.; Beatson, Scott A.; Ulett, Kimberly B.; Benjamin, William H.; Davies, Mark R.; Dando, Samantha J.; King, Nathan P.; Cripps, Allan W.; Dougan, Gordon

2015-01-01

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU. PMID:26553467

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6.

PubMed

Aoki, Y; Jones, K D; Tosato, G

2000-04-01

Since the discovery of the virus in 1994, the rapid pace with which Karposi's sarcoma-associated herpesvirus (KSHV) research has progressed has quickly led to a broad understanding of the structure of the virus and its biology and pathology in humans. Molecular piracy of potentially useful cellular genes has emerged as a characteristic feature of this virus. The viral homolog of human IL-6, vIL-6 is an example in kind. Studies in vitro and in vivo have shown that vIL-6 can stimulate the growth of KSHV-infected primary infusion lymphoma (PEL) cells, can promote hematopoiesis, and act as an angiogenic factor through the induction of vascular endothelial growth factor (VEGF). It is not difficult to envision how vIL-6, through these properties and perhaps others yet to be identified, can contribute to KSHV survival and spread in the human population.

Epstein–Barr virus latent genes

PubMed Central

Kang, Myung-Soo; Kieff, Elliott

2015-01-01

Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized. PMID:25613728

P-cadherin regulates human hair growth and cycling via canonical Wnt signaling and transforming growth factor-β2.

PubMed

Samuelov, Liat; Sprecher, Eli; Tsuruta, Daisuke; Bíró, Tamás; Kloepper, Jennifer E; Paus, Ralf

2012-10-01

P-cadherin is a key component of epithelial adherens junctions, and it is prominently expressed in the hair follicle (HF) matrix. Loss-of-function mutations in CDH3, which encodes P-cadherin, result in hypotrichosis with juvenile macular dystrophy (HJMD), an autosomal recessive disorder featuring sparse and short hair. Here, we attempted to recapitulate some aspects of HJMD in vitro by transfecting normal, organ-cultured human scalp HFs with lipofectamine and CDH3-specific or scrambled control siRNAs. As in HJMD patients, P-cadherin silencing inhibited hair shaft growth, prematurely induced HF regression (catagen), and inhibited hair matrix keratinocyte proliferation. In situ, membrane β-catenin expression and transcription of the β-catenin target gene, axin2, were significantly reduced, whereas glycogen synthase kinase 3 β (GSK3β) and phospho-β-catenin immunoreactivity were increased. These effects were partially reversed by inhibiting GSK3β. P-cadherin silencing reduced the expression of the anagen-promoting growth factor, IGF-1, whereas that of transforming growth factor β 2 (TGFβ2; catagen promoter) was enhanced. Neutralizing TGFβ antagonized the catagen-promoting effects of P-cadherin silencing. In summary, we introduce human HFs as an attractive preclinical model for studying the functions of P-cadherin in human epithelial biology and pathology. This model demonstrates that cadherins can be successfully knocked down in an intact human organ in vitro, and shows that P-cadherin is needed for anagen maintenance by regulating canonical Wnt signaling and suppressing TGFβ2.

Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals

PubMed Central

Theilmann, Mia C.; Nielsen, Kristian Fog; Klaenhammer, Todd R.

2017-01-01

ABSTRACT Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus. These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host. PMID:29162708

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer.

PubMed

Pera, Edgar M; Kim, James I; Martinez, Sarah L; Brechner, Mariel; Li, Su Yu; Wessely, Oliver; De Robertis, E M

2002-08-01

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.

Residue 41 of the Eurasian Avian-Like Swine Influenza A Virus Matrix Protein Modulates Virion Filament Length and Efficiency of Contact Transmission

PubMed Central

Campbell, Patricia J.; Kyriakis, Constantinos S.; Marshall, Nicolle; Suppiah, Suganthi; Seladi-Schulman, Jill; Danzy, Shamika; Lowen, Anice C.

2014-01-01

ABSTRACT Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity. IMPORTANCE We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model. PMID:24760887

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

PubMed

Ipe, Deepak S; Ben Zakour, Nouri L; Sullivan, Matthew J; Beatson, Scott A; Ulett, Kimberly B; Benjamin, William H; Davies, Mark R; Dando, Samantha J; King, Nathan P; Cripps, Allan W; Schembri, Mark A; Dougan, Gordon; Ulett, Glen C

2016-01-01

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

CDKN2B loss promotes progression from benign melanocytic nevus to melanoma

PubMed Central

McNeal, Andrew S.; Liu, Kevin; Nakhate, Vihang; Natale, Christopher A.; Duperret, Elizabeth K.; Capell, Brian C.; Dentchev, Tzvete; Berger, Shelley L.; Herlyn, Meenhard; Seykora, John T.; Ridky, Todd W.

2015-01-01

Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E) activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Further, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. PMID:26183406

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

PubMed

Zhu, Y; Lin, E C

1988-05-01

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

PubMed Central

Zhu, Y; Lin, E C

1988-01-01

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose. PMID:2834341

MicroRNA-627 Mediates the Epigenetic Mechanisms of Vitamin D to Suppress Proliferation of Human Colorectal Cancer Cells and Growth of Xenograft Tumors in Mice

PubMed Central

Padi, Sathish K.R.; Zhang, Qunshu; Rustum, Youcef M; Morrison, Carl; Guo, Bin

2013-01-01

Background & Aims Vitamin D protects against colorectal cancer by unclear mechanisms. We investigated the effects of calcitriol (1α,25-dihydroxyvitamin D3, the active form of vitamin D) on levels of different microRNAs (miRs) in colorectal cancer (CRC) cells from humans and xenograft tumors in mice. Methods Expression of microRNAs in CRC cell lines was examined using the Ambion mirVana miRNA Bioarray. The effects of calcitriol on expression of miR-627 and cell proliferation were determined by real-time PCR and WST-1 assay, respectively; growth of colorectal xenograft tumors was examined in nude mice. Real-time PCR was used to analyze levels of miR-627 in human colon adenocarcinoma samples and non-tumor colon mucosa tissues (controls). Results In HT-29 cells, miR-627 was the only microRNA significantly upregulated by calcitriol. Jumonji domain containing 1A (JMJD1A), which encodes a histone demethylase, was found to be a target of miR-627. By downregulating JMJD1A, miR-627 increased methylation of histone H3K9 and suppressed expression of proliferative factors such as GDF15. Calcitriol induced expression of miR-627, which downregulated JMJD1A and suppressed growth of xenograft tumors from HCT-116 cells in nude mice. Overexpression of miR-627 prevented proliferation of CRC cell lines in culture and growth of xenograft tumors in mice. Conversely, blocking the activity of miR-627 inhibited the tumor suppressive effects of calcitriol in cultured CRC cells and in mice. Levels of miR-627 were decreased in human colon adenocarcinoma samples, compared with controls. Conclusions miR-627 mediates tumor-suppressive epigenetic activities of vitamin D on CRC cells and xenograft tumors in mice. The mRNA that encodes the histone demethylase JMJD1A is a direct target of miR-627. Reagents designed to target JMJD1A or its mRNA, or increase the function of miR-627, might have the same antitumor activities of vitamin D without the hypercalcemic side effects. PMID:23619147

Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

PubMed Central

Silva, Maria C.; Yu, Qian-Chun; Enquist, Lynn; Shenk, Thomas

2003-01-01

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm. PMID:12970444

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

PubMed

Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

2008-07-01

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

A short synthetic peptide fragment of human C2ORF40 has therapeutic potential in breast cancer

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

Lin, Chaoyang; Zhang, Pengju; Jiang, Anli

C2ORF40 encodes a secreted protein which is cleaved to generate soluble peptides by proteolytic processing and this process is believed to be necessary for C2ORF40 to exert cell type specific biological activity. Here, we reported a short mimic peptide of human C2ORF40 acts potential therapeutic efficacy in human cancer cells in vitro and in vivo. We synthesized a short peptide of human C2ORF40, named C2ORF40 mimic peptide fragment and assessed its biological function on cancer cell growth, migration and tumorigenesis. Cell growth assay showed that C2ORF40 mimic peptide fragment significantly suppressed cell proliferation of breast and lung cancer cells. Moreover,more » C2ORF40 mimic peptide fragment significantly inhibited the migration and invasion of breast cancer cells. Furthermore, we showed that this peptide suppressed tumorigenesis in breast tumor xenograft model. Cell cycle assay indicated that the C2ORF40 mimic peptide fragment suppressed the growth of tumor cells through inducing mitotic phase arrest. In conclusion, our results firstly suggested that this short synthetic peptide of human C2ORF40 may be a candidate tumor therapeutic agent.« less

Non-hepatic tumors change the activity of genes encoding copper trafficking proteins in the liver

PubMed Central

Babich, Polina S.; Skvortsov, Alexey N; Rusconi, Paolo; Tsymbalenko, Nadezhda V.; Mutanen, Marja; Puchkova, Ludmila V.; Broggini, Massimo

2013-01-01

To assess the statistical relationship between tumor growth and copper metabolism, we performed a metaanalysis of studies in which patients with neoplasms were characterized according to any of the copper status indexes (atomic copper serum concentration, serum oxidase activity, ceruloplasmin protein content). Our metaanalysis shows that in the majority of cases (more than 3100 patients), tumor growth positively correlates with the copper status indexes. Nude athymic CD-1 nu/nu mice with subcutaneous tumors of human origin, C57Bl/6J mice with murine melanoma and ApcMin mice with spontaneously developing adenomas throughout the intestinal tract were studied to experimentally determine the relationship between tumor progression, liver copper metabolism, and copper status indexes. We showed that the copper status indexes increased significantly during tumor growth. In the liver tissue of tumor-bearing mice, ceruloplasmin gene expression, as well as the expression of genes related to ceruloplasmin metallation (CTR1 and ATP7B), increased significantly. Moreover, the presence of an mRNA splice variant encoding a form of ceruloplasmin anchored to the plasma membrane by glycosylphosphatidyl inositol, which is atypical for hepatocytes, was also detected. The ATP7A copper transporter gene, which is normally expressed in the liver only during embryonic copper metabolism, was also activated. Depletion of holo-ceruloplasmin resulted in retardation of human HCT116 colon carcinoma cell growth in nude mice and induced DNA fragmentation in tumor cells. In addition, the concentration of cytochrome c increased significantly in the cytosol, while decreasing in the mitochondria. We discuss a possible trans-effect of developing tumors on copper metabolism in the liver. PMID:23792645

Transgenic cells with increased plastoquinone levels and methods of use

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

Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

PubMed

Lee, Soo Chan; Phadke, Sujal; Sun, Sheng; Heitman, Joseph

2012-11-01

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.

An abundance of rare functional variants in 202 drug target genes sequenced in 14,002 people

PubMed Central

Nelson, Matthew R.; Wegmann, Daniel; Ehm, Margaret G.; Kessner, Darren; St. Jean, Pamela; Verzilli, Claudio; Shen, Judong; Tang, Zhengzheng; Bacanu, Silviu-Alin; Fraser, Dana; Warren, Liling; Aponte, Jennifer; Zawistowski, Matthew; Liu, Xiao; Zhang, Hao; Zhang, Yong; Li, Jun; Li, Yun; Li, Li; Woollard, Peter; Topp, Simon; Hall, Matthew D.; Nangle, Keith; Wang, Jun; Abecasis, Gonçalo; Cardon, Lon R.; Zöllner, Sebastian; Whittaker, John C.; Chissoe, Stephanie L.; Novembre, John; Mooser, Vincent

2015-01-01

Rare genetic variants contribute to complex disease risk; however, the abundance of rare variants in human populations remains unknown. We explored this spectrum of variation by sequencing 202 genes encoding drug targets in 14,002 individuals. We find rare variants are abundant (one every 17 bases) and geographically localized, such that even with large sample sizes, rare variant catalogs will be largely incomplete. We used the observed patterns of variation to estimate population growth parameters, the proportion of variants in a given frequency class that are putatively deleterious, and mutation rates for each gene. Overall we conclude that, due to rapid population growth and weak purifying selection, human populations harbor an abundance of rare variants, many of which are deleterious and have relevance to understanding disease risk. PMID:22604722

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

PubMed Central

Hsu, Cary; Jones, Stephanie A.; Cohen, Cyrille J.; Zheng, Zhili; Kerstann, Keith; Zhou, Juhua; Robbins, Paul F.; Peng, Peter D.; Shen, Xinglei; Gomes, Theotonius J.; Dunbar, Cynthia E.; Munroe, David J.; Stewart, Claudia; Cornetta, Kenneth; Wangsa, Danny; Ried, Thomas; Rosenberg, Steven A.

2007-01-01

Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation. PMID:17353346

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

PubMed

Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

2016-12-27

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( PCGEM1 , PEG3 , EPHA3 , and EFNB2 ) or other types of human cancers ( TOX3 , ST8SIA4 , and SLITRK3 ), and genes that are diagnostic/prognostic biomarkers of prostate cancer ( GRINA3 , and BCHE ).

Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

PubMed Central

Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

2014-01-01

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

Mechanisms and pathways of growth failure in primordial dwarfism.

PubMed

Klingseisen, Anna; Jackson, Andrew P

2011-10-01

The greatest difference between species is size; however, the developmental mechanisms determining organism growth remain poorly understood. Primordial dwarfism is a group of human single-gene disorders with extreme global growth failure (which includes Seckel syndrome, microcephalic osteodysplastic primordial dwarfism I [MOPD] types I and II, and Meier-Gorlin syndrome). Ten genes have now been identified for microcephalic primordial dwarfism, encoding proteins involved in fundamental cellular processes including genome replication (ORC1 [origin recognition complex 1], ORC4, ORC6, CDT1, and CDC6), DNA damage response (ATR [ataxia-telangiectasia and Rad3-related]), mRNA splicing (U4atac), and centrosome function (CEP152, PCNT, and CPAP). Here, we review the cellular and developmental mechanisms underlying the pathogenesis of these conditions and address whether further study of these genes could provide novel insight into the physiological regulation of organism growth.

The LKB1-AMPK pathway: metabolism and growth control in tumour suppression.

PubMed

Shackelford, David B; Shaw, Reuben J

2009-08-01

In the past decade, studies of the human tumour suppressor LKB1 have uncovered a novel signalling pathway that links cell metabolism to growth control and cell polarity. LKB1 encodes a serine-threonine kinase that directly phosphorylates and activates AMPK, a central metabolic sensor. AMPK regulates lipid, cholesterol and glucose metabolism in specialized metabolic tissues, such as liver, muscle and adipose tissue. This function has made AMPK a key therapeutic target in patients with diabetes. The connection of AMPK with several tumour suppressors suggests that therapeutic manipulation of this pathway using established diabetes drugs warrants further investigation in patients with cancer.

An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

PubMed

Jaquemar, D; Schenker, T; Trueb, B

1999-03-12

We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors.

PubMed

Galimi, F; Bagnara, G P; Bonsi, L; Cottone, E; Follenzi, A; Simeone, A; Comoglio, P M

1994-12-01

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c-MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU-GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization.

PubMed

Paterno, Gary D; Ding, Zhihu; Lew, Yuan-Y; Nash, Gord W; Mercer, F Corinne; Gillespie, Laura L

2002-07-24

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

PubMed Central

Sherman, Michael Y.; Gabai, Vladimir; Kiselev, Oleg; Komissarov, Andrey; Grudinin, Mikhail; Shartukova, Maria; Romanovskaya-Romanko, Ekaterina A.; Kudryavets, Yuri; Bezdenezhnykh, Natalya; Lykhova, Oleksandra; Semesyuk, Nadiia; Concetti, Antonio; Tsyb, Anatoly; Filimonova, Marina; Makarchuk, Victoria; Yakubovsky, Raisa; Chursov, Andrey; Shcherbinina, Vita; Shneider, Alexander

2013-01-01

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials. PMID:24121124

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia

PubMed Central

Smout, Michael J.; Sotillo, Javier; Laha, Thewarach; Papatpremsiri, Atiroch; Rinaldi, Gabriel; Pimenta, Rafael N.; Chan, Lai Yue; Johnson, Michael S.; Turnbull, Lynne; Whitchurch, Cynthia B.; Giacomin, Paul R.; Moran, Corey S.; Golledge, Jonathan; Daly, Norelle; Sripa, Banchob; Mulvenna, Jason P.

2015-01-01

Abstract Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world. PMID:26485648

Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element. | Office of Cancer Genomics

Cancer.gov

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius

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

Lee, Brady D.; Apel, William A.; DeVeaux, Linda C.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. A. acidocaldarius grew while simultaneouslymore » metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two. Regulation occurred primarily in genes: 1) encoding regulators, 2) encoding enzymes for cell synthesis, and 3) encoding sugar transporters.« less

Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23

PubMed Central

Bäuerl, Christine; Pérez-Martínez, Gaspar; Yan, Fang; Polk, D. Brent; Monedero, Vicente

2011-01-01

The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria PMID:21178363

The complete sequence and promoter activity of the human A-raf-1 gene (ARAF1)

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

Lee, J.E.; Beck, T.W.; Brennscheidt, U.

1994-03-01

The raf proto-oncogenes encode cytoplasmic protein serine/threonine kinases, which play a critical role in cell growth and development. One of these, A-raf-1 (human gene symbol, ARAF1), which is predominantly expressed in mouse urogenital tissues, has been mapped to an evolutionarily conserved linkage group composed of ARAF1, SYN1, TIMP, and properdin located at human chromosome Xp11.2. The authors have isolated human genomic DNA clones containing the expressed gene (ARAF1) on the X chromosome and a pseudogene (ARAF2) on chromosome 7p12-q11.21. Analysis of the nucleotide sequence from the ARAF1 genomic clones demonstrated that it consists of 16 exons encoded by minimally 10,776more » nucleotides. The major transcriptional start site (+1) was determined by RNase protection and primer extension assays. Promoter activity was confirmed by functional assays using DNA fragments fused to a CAT reporter gene. The ARAF1 minimal promoter, located between nucleotides -59 and +93, has a low G + C content and lacks consensus TATA and Inr sequences but shows sequence similarity at position -1 to the E box that is known to interact with USF and TFII-I transcription factors. 65 refs., 7 figs., 1 tab.« less

Impact assessment of bisphenol A on lignin-modifying enzymes by basidiomycete Trametes versicolor.

PubMed

Takamiya, Minako; Magan, Naresh; Warner, Philip J

2008-06-15

The impact of different concentrations of bisphenol A (BPA) was evaluated on growth of the white-rot basidiomycete, Trametes versicolor, and on the expression of genes encoding lignin-modifying enzyme (LME) activities. Effective doses (EDs) were obtained from fungal growth rate to monitor LME activities and the expression levels of their encoding genes. The fungus showed mycelial growth at concentrations of up to 300 microg ml(-1) of BPA with an ED50 value of 185 microg ml(-1). The LME activities were stimulated by BPA concentrations up to 300 microg ml(-1). The lignin peroxidase (LIP) encoding gene may be sensitive to BPA stress.

Mechanisms and pathways of growth failure in primordial dwarfism

PubMed Central

Klingseisen, Anna; Jackson, Andrew P.

2011-01-01

The greatest difference between species is size; however, the developmental mechanisms determining organism growth remain poorly understood. Primordial dwarfism is a group of human single-gene disorders with extreme global growth failure (which includes Seckel syndrome, microcephalic osteodysplastic primordial dwarfism I [MOPD] types I and II, and Meier-Gorlin syndrome). Ten genes have now been identified for microcephalic primordial dwarfism, encoding proteins involved in fundamental cellular processes including genome replication (ORC1 [origin recognition complex 1], ORC4, ORC6, CDT1, and CDC6), DNA damage response (ATR [ataxia-telangiectasia and Rad3-related]), mRNA splicing (U4atac), and centrosome function (CEP152, PCNT, and CPAP). Here, we review the cellular and developmental mechanisms underlying the pathogenesis of these conditions and address whether further study of these genes could provide novel insight into the physiological regulation of organism growth. PMID:21979914

Generation of fibrosarcomas in vivo by a retrovirus that expresses the normal B chain of platelet-derived growth factor and mimics the alternative splice pattern of the v-sis oncogene

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

Pech, M.; Gazit, A.; Arnstein, P.

1989-04-01

A retrovirus containing the entire human platelet-derived growth factor B-chain (PDGF-B) gene was constructed in order to investigate the in vivo biological activity of its encoded growth factor. When this virus was introduced into newborn mice, it reproducibly generated fibrosarcomas at the site of inoculation. Proviruses in each fibrosarcoma analyzed had lost 149 nucleotides downstream of the PDGF-B coding region. This deletion originated from an alternative or aberrant splice event that occurred within exon 7 of the PDGF-B gene and mimicked the v-sis oncogene. Thus, deletion of this region may be necessary for efficient retrovirus replication or for more potentmore » transforming function. Evidence that the normal growth factor coding sequence was unaltered derived from RNase protection studies and immunoprecipitation analysis. Tumors were generally polyclonal but demonstrated clonal subpopulations. Moreover, tumor-derived cell lines became monoclonal within a few tissue culture passages and rapidly formed tumors in vivo. These findings argue that overexpression of the normal human PDGF-B gene product under retrovirus control can induce the fully malignant phenotype.« less

Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

PubMed

Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

2014-09-02

Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

The Moraxella catarrhalis nitric oxide reductase is essential for nitric oxide detoxification.

PubMed

Wang, Wei; Kinkel, Traci; Martens-Habbena, Willm; Stahl, David A; Fang, Ferric C; Hansen, Eric J

2011-06-01

Moraxella catarrhalis is a Gram-negative obligate aerobe that is an important cause of human respiratory tract infections. The M. catarrhalis genome encodes a predicted truncated denitrification pathway that reduces nitrate to nitrous oxide. We have previously shown that expression of both the M. catarrhalis aniA (encoding a nitrite reductase) and norB (encoding a putative nitric oxide reductase) genes is repressed by the transcriptional regulator NsrR under aerobic conditions and that M. catarrhalis O35E nsrR mutants are unable to grow in the presence of low concentrations of nitrite (W. Wang, et al., J. Bacteriol. 190:7762-7772, 2008). In this study, we constructed an M. catarrhalis norB mutant and showed that planktonic growth of this mutant is inhibited by low levels of nitrite, whether or not an nsrR mutation is present. To determine the importance of NorB in this truncated denitrification pathway, we analyzed the metabolism of nitrogen oxides by norB, aniA norB, and nsrR norB mutants. We found that norB mutants are unable to reduce nitric oxide and produce little or no nitrous oxide from nitrite. Furthermore, nitric oxide produced from nitrite by the AniA protein is bactericidal for a Moraxella catarrhalis O35E norB mutant but not for wild-type O35E bacteria under aerobic growth conditions in vitro, suggesting that nitric oxide catabolism in M. catarrhalis is accomplished primarily by the norB gene product. Measurement of bacterial protein S-nitrosylation directly implicates nitrosative stress resulting from AniA-dependent nitric oxide formation as a cause of the growth inhibition of norB and nsrR mutants by nitrite.

Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function

PubMed Central

Han, Li; Szabó, Piroska E.; Mann, Jeffrey R.

2010-01-01

The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome. PMID:20062522

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

PubMed

Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

2018-04-01

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

PubMed Central

Hu, Patrick J; Xu, Jinling; Ruvkun, Gary

2006-01-01

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues. PMID:16839187

Mutations in C4orf26, encoding a peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta.

PubMed

Parry, David A; Brookes, Steven J; Logan, Clare V; Poulter, James A; El-Sayed, Walid; Al-Bahlani, Suhaila; Al Harasi, Sharifa; Sayed, Jihad; Raïf, El Mostafa; Shore, Roger C; Dashash, Mayssoon; Barron, Martin; Morgan, Joanne E; Carr, Ian M; Taylor, Graham R; Johnson, Colin A; Aldred, Michael J; Dixon, Michael J; Wright, J Tim; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2012-09-07

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Decoding the disease-associated proteins encoded in the human chromosome 4.

PubMed

Chen, Lien-Chin; Liu, Mei-Ying; Hsiao, Yung-Chin; Choong, Wai-Kok; Wu, Hsin-Yi; Hsu, Wen-Lian; Liao, Pao-Chi; Sung, Ting-Yi; Tsai, Shih-Feng; Yu, Jau-Song; Chen, Yu-Ju

2013-01-04

Chromosome 4 is the fourth largest chromosome, containing approximately 191 megabases (~6.4% of the human genome) with 757 protein-coding genes. A number of marker genes for many diseases have been found in this chromosome, including genetic diseases (e.g., hepatocellular carcinoma) and biomedical research (cardiac system, aging, metabolic disorders, immune system, cancer and stem cell) related genes (e.g., oncogenes, growth factors). As a pilot study for the chromosome 4-centric human proteome project (Chr 4-HPP), we present here a systematic analysis of the disease association, protein isoforms, coding single nucleotide polymorphisms of these 757 protein-coding genes and their experimental evidence at the protein level. We also describe how the findings from the chromosome 4 project might be used to drive the biomarker discovery and validation study in disease-oriented projects, using the examples of secretomic and membrane proteomic approaches in cancer research. By integrating with cancer cell secretomes and several other existing databases in the public domain, we identified 141 chromosome 4-encoded proteins as cancer cell-secretable/shedable proteins. Additionally, we also identified 54 chromosome 4-encoded proteins that have been classified as cancer-associated proteins with successful selected or multiple reaction monitoring (SRM/MRM) assays developed. From literature annotation and topology analysis, 271 proteins were recognized as membrane proteins while 27.9% of the 757 proteins do not have any experimental evidence at the protein-level. In summary, the analysis revealed that the chromosome 4 is a rich resource for cancer-associated proteins for biomarker verification projects and for drug target discovery projects.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

PubMed

Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Walpole, Carina M; Maugham, Michelle; Fung, Jenny N T; Yap, Pei-Yi; O'Keeffe, Angela J; Lai, John; Whiteside, Eliza J; Herington, Adrian C; Chopin, Lisa K

2016-06-01

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells.

PubMed

Chen, Z; Chen, J; Gu, Y; Hu, C; Li, J-L; Lin, S; Shen, H; Cao, C; Gao, R; Li, J; Ha, P K; Kaye, F J; Griffin, J D; Wu, L

2014-07-17

Salivary gland tumors (SGT) are a group of highly heterogeneous head and neck malignancies with widely varied clinical outcomes and no standard effective treatments. The CRTC1-MAML2 fusion oncogene, encoded by a recurring chromosomal translocation t(11;19)(q14-21;p12-13), is a frequent genetic alteration found in >50% of mucoepidermoid carcinomas (MEC), the most common malignant SGT. In this study, we aimed to define the role of the CRTC1-MAML2 oncogene in the maintenance of MEC tumor growth and to investigate critical downstream target genes and pathways for therapeutic targeting of MEC. By performing gene expression analyses and functional studies via RNA interference and pharmacological modulation, we determined the importance of the CRTC1-MAML2 fusion gene and its downstream AREG-EGFR signaling in human MEC cancer cell growth and survival in vitro and in vivo using human MEC xenograft models. We found that CRTC1-MAML2 fusion oncogene was required for the growth and survival of fusion-positive human MEC cancer cells in vitro and in vivo. The CRTC1-MAML2 oncoprotein induced the upregulation of the epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) by co-activating the transcription factor CREB, and AREG subsequently activated EGFR signaling in an autocrine manner that promoted MEC cell growth and survival. Importantly, CRTC1-MAML2-positive MEC cells were highly sensitive to EGFR signaling inhibition. Therefore, our study revealed that aberrantly activated AREG-EGFR signaling is required for CRTC1-MAML2-positive MEC cell growth and survival, suggesting that EGFR-targeted therapies will benefit patients with advanced, unresectable CRTC1-MAML2-positive MEC.

POC1A truncation mutation causes a ciliopathy in humans characterized by primordial dwarfism.

PubMed

Shaheen, Ranad; Faqeih, Eissa; Shamseldin, Hanan E; Noche, Ramil R; Sunker, Asma; Alshammari, Muneera J; Al-Sheddi, Tarfa; Adly, Nouran; Al-Dosari, Mohammed S; Megason, Sean G; Al-Husain, Muneera; Al-Mohanna, Futwan; Alkuraya, Fowzan S

2012-08-10

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility

PubMed Central

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A.; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M.; Stracker, Travis H.; Valverde, Miguel A.; Muñoz, Francisco J.; Gil-Gómez, Gabriel

2017-01-01

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients. PMID:29245899

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

PubMed

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M; Stracker, Travis H; Valverde, Miguel A; Muñoz, Francisco J; Gil-Gómez, Gabriel

2017-11-21

Cyclin O (encoded by CCNO ) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo , we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno -/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno +/- mice also developed hydrocephalus and affected Ccno -/- and Ccno +/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno -/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.

PubMed

Jia, Yimin; Song, Haogang; Gao, Guichao; Cai, Demin; Yang, Xiaojing; Zhao, Ruqian

2015-11-25

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.

Amphetamine fails to alter cued recollection of emotional images: study of encoding, retrieval, and state-dependency.

PubMed

Weafer, Jessica; Gallo, David A; de Wit, Harriet

2014-01-01

Stimulant drugs facilitate both encoding and retrieval of salient information in laboratory animals, but less is known about their effects on memory for emotionally salient visual images in humans. The current study investigated dextroamphetamine (AMP) effects on memory for emotional pictures in healthy humans, by administering the drug only at encoding, only at retrieval, or at both encoding and retrieval. During the encoding session, all participants viewed standardized positive, neutral, and negative pictures from the International Affective Picture System (IAPS). 48 hours later they attended a retrieval session testing their cued recollection of these stimuli. Participants were randomly assigned to one of four conditions (N=20 each): condition AP (20 mg AMP at encoding and placebo (PL) at retrieval); condition PA (PL at encoding and AMP at retrieval); condition AA (AMP at encoding and retrieval); or condition PP (PL at encoding and retrieval). Amphetamine produced its expected effects on physiological and subjective measures, and negative pictures were recollected more frequently than neutral pictures. However, contrary to hypotheses, AMP did not affect recollection for positive, negative, or neutral stimuli, whether it was administered at encoding, retrieval, or at both encoding and retrieval. Moreover, recollection accuracy was not state-dependent. Considered in light of other recent drug studies in humans, this study highlights the sensitivity of drug effects to memory testing conditions and suggests future strategies for translating preclinical findings to human behavioral laboratories.

Amphetamine Fails to Alter Cued Recollection of Emotional Images: Study of Encoding, Retrieval, and State-Dependency

PubMed Central

Weafer, Jessica; Gallo, David A.; de Wit, Harriet

2014-01-01

Stimulant drugs facilitate both encoding and retrieval of salient information in laboratory animals, but less is known about their effects on memory for emotionally salient visual images in humans. The current study investigated dextroamphetamine (AMP) effects on memory for emotional pictures in healthy humans, by administering the drug only at encoding, only at retrieval, or at both encoding and retrieval. During the encoding session, all participants viewed standardized positive, neutral, and negative pictures from the International Affective Picture System (IAPS). 48 hours later they attended a retrieval session testing their cued recollection of these stimuli. Participants were randomly assigned to one of four conditions (N = 20 each): condition AP (20 mg AMP at encoding and placebo (PL) at retrieval); condition PA (PL at encoding and AMP at retrieval); condition AA (AMP at encoding and retrieval); or condition PP (PL at encoding and retrieval). Amphetamine produced its expected effects on physiological and subjective measures, and negative pictures were recollected more frequently than neutral pictures. However, contrary to hypotheses, AMP did not affect recollection for positive, negative, or neutral stimuli, whether it was administered at encoding, retrieval, or at both encoding and retrieval. Moreover, recollection accuracy was not state-dependent. Considered in light of other recent drug studies in humans, this study highlights the sensitivity of drug effects to memory testing conditions and suggests future strategies for translating preclinical findings to human behavioral laboratories. PMID:24587355

Streptococcus pneumoniae Can Utilize Multiple Sources of Hyaluronic Acid for Growth

PubMed Central

Marion, Carolyn; Stewart, Jason M.; Tazi, Mia F.; Burnaugh, Amanda M.; Linke, Caroline M.; Woodiga, Shireen A.

2012-01-01

The mechanisms by which Streptococcus pneumoniae obtains carbohydrates for growth during airway colonization remain to be elucidated. The low concentration of free carbohydrates in the normal human airway suggests that pneumococci must utilize complex glycan structures for growth. The glycosaminoglycan hyaluronic acid is present on the apical surface of airway epithelial cells. As pneumococci express a hyaluronate lyase (Hyl) that cleaves hyaluronic acid into disaccharides, we hypothesized that during colonization pneumococci utilize the released carbohydrates for growth. Hyaluronic acid supported significant pneumococcal growth in an hyl-dependent manner. A phosphoenolpyruvate-dependent phosphotransferase system (PTS) and an unsaturated glucuronyl hydrolase (Ugl) encoded downstream of hyl are also essential for growth on hyaluronic acid. This genomic arrangement is present in several other organisms, suggesting conservation of the utilization mechanism between species. In vivo experiments support the hypothesis that S. pneumoniae utilizes hyaluronic acid as a carbon source during colonization. We also demonstrate that pneumococci can utilize the hyaluronic acid capsule of other bacterial species for growth, suggesting an alternative carbohydrate source for pneumococcal growth. Together, these data support a novel function for pneumococcal degradation of hyaluronic acid in vivo and provide mechanistic details of growth on this glycosaminoglycan. PMID:22311922

Expression, purification, and lipolytic activity of recombinant human serum albumin fusion proteins with one domain of human growth hormone in Pichia pastoris.

PubMed

Wang, Furong; Wu, Min; Liu, Wenhui; Shen, Qi; Sun, Hongying; Chen, Shuqing

2013-01-01

Human growth hormone (hGH) can mobilize lipid and inhibit the synthesis of triglycerides. However, it is not a potentially useful drug for treating obesity because it has many other actions resulting in several side effects. Here, we report a novel approach to develop the lipolytic function of hGH. The amino terminus of hGH was replaced by an inactive protein so that the actions unrelated to lipolytic function would be avoided. The fusion genes encoding human serum albumin (HSA) and lipolytic domain of hGH were constructed and expressed in Pichia pastoris. The recombinant proteins were purified and characterized by SDS-PAGE and Western blot. The preliminary stability tests demonstrated that HSA-hGH166-191 and HSA-hGH177-191 were stable at different pH levels after four days at 37°C. Lipolytic activity assay revealed that fusion proteins could increase the amounts of glycerol released from the isolated adipocytes. The HSA fusion proteins constructed in this work can be further developed as antiobesity agents. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

PubMed

Blázquez, M A; Gamo, F J; Gancedo, C

1995-12-18

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Human Genomic Signatures of Brain Oscillations During Memory Encoding.

PubMed

Berto, Stefano; Wang, Guang-Zhong; Germi, James; Lega, Bradley C; Konopka, Genevieve

2018-05-01

Memory encoding is an essential step for all learning. However, the genetic and molecular mechanisms underlying human memory encoding remain poorly understood, and how this molecular framework permits the emergence of specific patterns of brain oscillations observed during mnemonic processing is unknown. Here, we directly compare intracranial electroencephalography recordings from the neocortex in individuals performing an episodic memory task with human gene expression from the same areas. We identify genes correlated with oscillatory memory effects across 6 frequency bands. These genes are enriched for autism-related genes and have preferential expression in neurons, in particular genes encoding synaptic proteins and ion channels, supporting the idea that the genes regulating voltage gradients are involved in the modulation of oscillatory patterns during successful memory encoding across brain areas. Memory-related genes are distinct from those correlated with other forms of cognitive processing and resting state fMRI. These data are the first to identify correlations between gene expression and active human brain states as well as provide a molecular window into memory encoding oscillations in the human brain.

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

PubMed

Wice, B M; Gordon, J I

1992-01-01

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal airway growth in cystic fibrosis piglets.

PubMed

Adam, Ryan J; Abou Alaiwa, Mahmoud H; Bouzek, Drake C; Cook, Daniel P; Gansemer, Nicholas D; Taft, Peter J; Powers, Linda S; Stroik, Mallory R; Hoegger, Mark J; McMenimen, James D; Hoffman, Eric A; Zabner, Joseph; Welsh, Michael J; Meyerholz, David K; Stoltz, David A

2017-09-01

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis. NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis. Copyright © 2017 the American Physiological Society.

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Speiseder, Thomas; Nevels, Michael; Dobner, Thomas

2014-01-01

The last 50 years of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells, human amniotic fluid cells and athymic nude mice.

Modulated Expression of Genes Encoding Estrogen Metabolizing Enzymes by G1-Phase Cyclin-Dependent Kinases 6 and 4 in Human Breast Cancer Cells

PubMed Central

Jia, Yi; Domenico, Joanne; Swasey, Christina; Wang, Meiqin; Gelfand, Erwin W.; Lucas, Joseph J.

2014-01-01

G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers. PMID:24848372

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

PubMed Central

Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

2016-01-01

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

PubMed Central

2011-01-01

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

PubMed Central

Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

1994-01-01

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

Modeling invasive breast cancer: growth factors propel progression of HER2-positive premalignant lesions

PubMed Central

Pradeep, C-R; Zeisel, A; Köstler, WJ; Lauriola, M; Jacob-Hirsch, J; Haibe-Kains, B; Amariglio, N; Ben-Chetrit, N; Emde, A; Solomonov, I; Neufeld, G; Piccart, M; Sagi, I; Sotiriou, C; Rechavi, G; Domany, E; Desmedt, C; Yarden, Y

2013-01-01

The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients’ lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment. PMID:22139081

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

Identification and Differential Abundance of Mitochondrial Genome Encoding Small RNAs (mitosRNA) in Breast Muscles of Modern Broilers and Unselected Chicken Breed

PubMed Central

Bottje, Walter G.; Khatri, Bhuwan; Shouse, Stephanie A.; Seo, Dongwon; Mallmann, Barbara; Orlowski, Sara K.; Pan, Jeonghoon; Kong, Seongbae; Owens, Casey M.; Anthony, Nicholas B.; Kim, Jae K.; Kong, Byungwhi C.

2017-01-01

Background: Although small non-coding RNAs are mostly encoded by the nuclear genome, thousands of small non-coding RNAs encoded by the mitochondrial genome, termed as mitosRNAs were recently reported in human, mouse and trout. In this study, we first identified chicken mitosRNAs in breast muscle using small RNA sequencing method and the differential abundance was analyzed between modern pedigree male (PeM) broilers (characterized by rapid growth and large muscle mass) and the foundational Barred Plymouth Rock (BPR) chickens (characterized by slow growth and small muscle mass). Methods: Small RNA sequencing was performed with total RNAs extracted from breast muscles of PeM and BPR (n = 6 per group) using the 1 × 50 bp single end read method of Illumina sequencing. Raw reads were processed by quality assessment, adapter trimming, and alignment to the chicken mitochondrial genome (GenBank Accession: X52392.1) using the NGen program. Further statistical analyses were performed using the JMP Genomics 8. Differentially expressed (DE) mitosRNAs between PeM and BPR were confirmed by quantitative PCR. Results: Totals of 183,416 unique small RNA sequences were identified as potential chicken mitosRNAs. After stringent filtering processes, 117 mitosRNAs showing >100 raw read counts were abundantly produced from all 37 mitochondrial genes (except D-loop region) and the length of mitosRNAs ranged from 22 to 46 nucleotides. Of those, abundance of 44 mitosRNAs were significantly altered in breast muscles of PeM compared to those of BPR: all mitosRNAs were higher in PeM breast except those produced from 16S-rRNA gene. Possibly, the higher mitosRNAs abundance in PeM breast may be due to a higher mitochondrial content compared to BPR. Our data demonstrate that in addition to 37 known mitochondrial genes, the mitochondrial genome also encodes abundant mitosRNAs, that may play an important regulatory role in muscle growth via mitochondrial gene expression control. PMID:29104541

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources.

PubMed

Khajanchi, Bijay K; Hasan, Nur A; Choi, Seon Young; Han, Jing; Zhao, Shaohua; Colwell, Rita R; Cerniglia, Carl E; Foley, Steven L

2017-08-02

The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

PubMed Central

Shaheen, Ranad; Faqeih, Eissa; Shamseldin, Hanan E.; Noche, Ramil R.; Sunker, Asma; Alshammari, Muneera J.; Al-Sheddi, Tarfa; Adly, Nouran; Al-Dosari, Mohammed S.; Megason, Sean G.; Al-Husain, Muneera; Al-Mohanna, Futwan; Alkuraya, Fowzan S.

2012-01-01

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein. PMID:22840364

RETREG1 (FAM134B): A new player in human diseases: 15 years after the discovery in cancer.

PubMed

Islam, Farhadul; Gopalan, Vinod; Lam, Alfred King-Yin

2018-06-01

FAM134B (family with sequence similarity 134, member B)/RETREG1 and its functional roles are relatively new in human diseases. This review aimed to summarize various functions of FAM134B since our first discovery of the gene in 2001. The protein encoded by FAM134B is a reticulophagy receptor that regulates turnover of the endoplasmic reticulum (ER) by selective phagocytosis. Absence or non-functional expression of FAM134B protein impairs ER-turnover and thereby is involved in the pathogenesis of some human diseases. FAM134B inhibition contributes to impair proteostasis in the ER due to the accumulation of misfolded or aggregated proteins, which in turn leads to compromised neuronal survival and progressive neuronal degenerative diseases. Mutations in FAM134B associated with hereditary sensory and autonomic neuropathy type IIB (HSAN IIB). Selective cleavage of FAM134B by Dengue, Zika, and West Nile virus encoded protease NS2B3 leads to the increased production of infection units, whereas upregulation of FAM134B inhibits viral replication. In cancer, FAM134B acts as a tumor suppressor and inhibit cancer growth both in-vitro and in-vivo. Pharmacological upregulation of FAM134B resulted in reduced cancer cell growth and proliferation. In addition, FAM134B mutations are common in patients with colorectal adenocarcinoma, and oesophageal squamous cell carcinoma. These mutations and expression changes of FAM134B were associated with the biological aggressiveness of these cancers. FAM134B also plays a role in allergic rhinitis, vascular dementia, and identification of stem cells. Taken together, information available in the literature suggests that FAM134B plays critical roles in human diseases, by interacting with different biological and chemical mediators, which are primarily regulated by ER turnover. © 2017 Wiley Periodicals, Inc.

Molecular cloning and characterization of chitinase genes from Candida albicans.

PubMed

McCreath, K J; Specht, C A; Robbins, P W

1995-03-28

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

Characterization of a highly conserved human homolog to the chicken neural cell surface protein Bravo/Nr-CAM that maps to chromosome band 7q31

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

Lane, R.P.; Vielmetter, J.; Dreyer, W.J.

1996-08-01

The neuronal cell adhesion molecule Bravo/Nr-CAM is a cell surface protein of the immunoglobulin (Ig) superfamily and is closely related to the L1/NgCAM and neurofascin molecules, all of which contain six immunoglobulin domains, five fibronectin repeats, a transmembrane region, and an intracellular domain. Chicken Bravo/Nr-CAM has been shown to interact with other cell surface molecules of the Ig superfamily and has been implicated in specific pathfinding roles of axonal growth cones in the developing nervous system. We now report the characterization of cDNA clones encoding the human Bravo/Nr-CAM protein, which, like its chicken homolog, is composed of six V-like Igmore » domains and five fibronectin type III repeats. The human Bravo/Nr-CAM homolog also contains a transmembrane and intracellular domain, both of which are 100% conserved at the amino acid level compared to its chicken homolog. Overall, the human Bravo/Nr-CAM homolog is 82% identical to the chicken Bravo/Nr-CAM amino acid sequence. Independent cDNAs encoding four different isoforms were also identified, all of which contain alternatively spliced variants around the fifth fibronectin type III repeat, including one isoform that had been previously identified for chicken Bravo/Nr-CAM. Northern blot analysis reveals one mRNA species of approximately 7.0 kb in adult human brain tissue. Fluorescence in situ hybridization maps the gene for human Bravo/Nr-CAM to human chromosome 7q31.1-q31.2. This chromosomal locus has been previously identified as containing a tumore suppressor candidate gene commonly deleted in certain human cancer tissues. 38 refs., 5 figs.« less

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

PubMed

Barrow, Alexander D; Edeling, Melissa A; Trifonov, Vladimir; Luo, Jingqin; Goyal, Piyush; Bohl, Benjamin; Bando, Jennifer K; Kim, Albert H; Walker, John; Andahazy, Mary; Bugatti, Mattia; Melocchi, Laura; Vermi, William; Fremont, Daved H; Cox, Sarah; Cella, Marina; Schmedt, Christian; Colonna, Marco

2018-01-25

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRβ signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning, overexpression, purification of bacteriocin enterocin-B and structural analysis, interaction determination of enterocin-A, B against pathogenic bacteria and human cancer cells.

PubMed

Ankaiah, Dasari; Palanichamy, Esakkiraj; Antonyraj, Christian Bharathi; Ayyanna, Repally; Perumal, Venkatesh; Ahamed, Syed Ibrahim Basheer; Arul, Venkatesan

2018-05-02

In this present study, a gene (ent-B) encoding the bacteriocin enterocin-B was cloned, overexpressed and purified from Enterococcus faecium por1. The molecular weight of the bacteriocin enterocin-B was observed around 7.2 kDa and exhibited antimicrobial activity against several human pathogenic bacteria. The antimicrobial activity of cloned enterocin-B was increased effectively by combining with another bacteriocin enterocin-A from the same microorganism. Protein-protein docking and molecular dynamics simulation studies revealed that the bacteriocin enterocin-B is interacting with enterocin-A and formation of a heterodimer (enterocin A + B). The heterodimer of bacteriocin enterocin-A + B exhibited potential anti-bacterial, anti-biofilm activity against Staphylococcus aureus, Acinetobacter baumannii, Listeria monocytogenes and Escherichia coli. The bacteriocin enterocin-B, A and heterodimer of bacteriocin enterocin A + B showed no haemolysis on human RBC cells. This is the first report that the cell growth inhibitory activity of the bacteriocin enterocin B against HeLa, HT-29 and AGS human cancer cells and this cell growth inhibitory activity was significantly increased when cancer cells treated with the heterodimer of bacteriocins enterocin-A + B. The cell growth inhibitory activity of the bacteriocin enterocin-B and the heterodimer of bacteriocin enterocin-A + B were not observed in non-cancerous INT-407 cells (intestinal epithelial cells). Copyright © 2018. Published by Elsevier B.V.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

DGK1-encoded Diacylglycerol Kinase Activity Is Required for Phospholipid Synthesis during Growth Resumption from Stationary Phase in Saccharomyces cerevisiae*

PubMed Central

Fakas, Stylianos; Konstantinou, Chrysanthos; Carman, George M.

2011-01-01

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity. PMID:21071438

Nitric Oxide Metabolism in Neisseria meningitidis

PubMed Central

Anjum, Muna F.; Stevanin, Tânia M.; Read, Robert C.; Moir, James W. B.

2002-01-01

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c′ are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress. PMID:12003939

p53 functions as a cell cycle control protein in osteosarcomas.

PubMed

Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-11-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

PubMed

Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

2000-11-10

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. Copyright 2000 Academic Press.

Perlecan and vascular endothelial growth factor-encoding DNA-loaded chitosan scaffolds promote angiogenesis and wound healing.

PubMed

Lord, Megan S; Ellis, April L; Farrugia, Brooke L; Whitelock, John M; Grenett, Hernan; Li, Chuanyu; O'Grady, Robert L; DeCarlo, Arthur A

2017-03-28

The repair of dermal wounds, particularly in the diabetic population, poses a significant healthcare burden. The impaired wound healing of diabetic wounds is attributed to low levels of endogenous growth factors, including vascular endothelial growth factor (VEGF), that normally stimulate multiple phases of wound healing. In this study, chitosan scaffolds were prepared via freeze drying and loaded with plasmid DNA encoding perlecan domain I and VEGF189 and analyzed in vivo for their ability to promote dermal wound healing. The plasmid DNA encoding perlecan domain I and VEGF189 loaded scaffolds promoted dermal wound healing in normal and diabetic rats. This treatment resulted in an increase in the number of blood vessels and sub-epithelial connective tissue matrix components within the wound beds compared to wounds treated with chitosan scaffolds containing control DNA or wounded controls. These results suggest that chitosan scaffolds containing plasmid DNA encoding VEGF189 and perlecan domain I have the potential to induce angiogenesis and wound healing. Copyright © 2017 Elsevier B.V. All rights reserved.

A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania

PubMed Central

Lyda, Todd A.; Joshi, Manju B.; Andersen, John F.; Kelada, Andrew Y.; Owings, Joshua P.; Bates, Paul A.; Dwyer, Dennis M.

2015-01-01

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts. PMID:25763714

A unique, highly conserved secretory invertase is differentially expressed by promastigote developmental forms of all species of the human pathogen, Leishmania.

PubMed

Lyda, Todd A; Joshi, Manju B; Andersen, John F; Kelada, Andrew Y; Owings, Joshua P; Bates, Paul A; Dwyer, Dennis M

2015-06-01

Leishmania are protozoan pathogens of humans that exist as extracellular promastigotes in the gut of their sand fly vectors and as obligate intracellular amastigotes within phagolysosomes of infected macrophages. Between infectious blood meal feeds, sand flies take plant juice meals that contain sucrose and store these sugars in their crop. Such sugars are regurgitated into the sand fly anterior midgut where they impact the developing promastigote parasite population. In this report we showed that promastigotes of all Leishmania species secreted an invertase/sucrase enzyme during their growth in vitro. In contrast, neither L. donovani nor L. mexicana amastigotes possessed any detectable invertase activity. Importantly, no released/secreted invertase activity was detected in culture supernatants from either Trypanosoma brucei or Trypanosoma cruzi. Using HPLC, the L. donovani secretory invertase was isolated and subjected to amino acid sequencing. Subsequently, we used a molecular approach to identify the LdINV and LmexINV genes encoding the ~72 kDa invertases produced by these organisms. Interestingly, we identified high fidelity LdINV-like homologs in the genomes of all Leishmania sp. but none were present in either T. brucei or T. cruzi. Northern blot and RT-PCR analyses showed that these genes were developmentally/differentially expressed in promastigotes but not amastigotes of these parasites. Homologous transfection studies demonstrated that these genes in fact encoded the functional secretory invertases produced by these parasites. Cumulatively, our results suggest that these secretory enzymes play critical roles in the survival/growth/development and transmission of all Leishmania parasites within their sand fly vector hosts.

Bone morphogenetic protein and activin membrane-bound inhibitor overexpression inhibits gastric tumor cell invasion via the transforming growth factor-β/epithelial-mesenchymal transition signaling pathway.

PubMed

Yuan, Chun-Ling; Liang, Rong; Liu, Zhi-Hui; Li, Yong-Qiang; Luo, Xiao-Ling; Ye, Jia-Zhou; Lin, Yan

2018-06-01

Gastric carcinoma is one of the most common human malignancies and remains the second leading cause of cancer-associated mortality worldwide. Gastric carcinoma is characterized by early-stage metastasis and is typically diagnosed in the advanced stage. Previous results have indicated that bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) overexpression has been demonstrated to inhibit growth and metastasis of gastric cancer cells. However, the molecular mechanisms of the BAMBI-mediated signaling pathway in the progression of gastric cancer are poorly understood. In the present study, to assess whether BAMBI overexpression inhibited the growth and aggressiveness of gastric carcinoma cells through regulation of transforming growth factor (TGF)-β/epithelial-mesenchymal transition (EMT) signaling pathway, the growth and metastasis of gastric carcinoma cells were analyzed following BAMBI overexpression and knockdown in vitro and in vivo . Molecular changes in the TGF-β/EMT signaling pathway were studied in gastric carcinoma cells following BAMBI overexpression and knockdown. DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway was investigated in gastric carcinoma cells. Tumor growth in tumor-bearing mice was analyzed after mice were subjected to endogenous overexpression of BAMBI. Results indicated that BAMBI overexpression significantly inhibited gastric carcinoma cell growth and aggressiveness, whereas knockdown of BAMBI significantly promoted its growth and metastasis compared with the control (P<0.01). The TGF-β/EMT signaling pathway was downregulated in BAMBI-overexpressed gastric carcinoma cells; however, signaling was promoted following BAMBI knockdown. In addition, it was observed that BAMBI overexpression significantly downregulated the DNA methylation of the gene regions encoding the TGF-β/EMT signaling pathway (P<0.01). Furthermore, RNA interference-mediated BAMBI overexpression also promoted apoptosis in gastric cancer cells and significantly inhibited growth of gastric tumors in murine xenografts (P<0.01). In conclusion, the present findings suggest that BAMBI overexpression inhibited the TGF-β/EMT signaling pathway and suppressed the invasiveness of gastric tumors, suggesting BAMBI may be a potential target for the treatment of gastric carcinoma via regulation of the TGF-β/EMT signaling pathway.

Drosophila mutants of the autism candidate gene neurobeachin (rugose) exhibit neuro-developmental disorders, aberrant synaptic properties, altered locomotion, and impaired adult social behavior and activity patterns.

PubMed

Wise, Alexandria; Tenezaca, Luis; Fernandez, Robert W; Schatoff, Emma; Flores, Julian; Ueda, Atsushi; Zhong, Xiaotian; Wu, Chun-Fang; Simon, Anne F; Venkatesh, Tadmiri

2015-01-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions, and hyperactivity. ASD exhibits a strong genetic component with underlying multigene interactions. Candidate gene studies have shown that the neurobeachin (NBEA) gene is disrupted in human patients with idiopathic autism ( Castermans et al., 2003 ). The NBEA gene spans the common fragile site FRA 13A and encodes a signal scaffold protein ( Savelyeva et al., 2006 ). In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. ( Medrihan et al., 2009 ; Savelyeva et al., 2006 ). Rugose (rg) is the Drosophila homolog of the mammalian and human NBEA. Our previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the epidermal growth factor receptor or EGFR and Notch-mediated signaling pathways, facilitating cross talk between these and other pathways ( Shamloula et al., 2002 ). We now present functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion, and hyperactivity. These results demonstrate that Drosophila NBEA (rg) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying ASDs.

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

PubMed Central

Selfors, Laura M.; Schutzman, Jennifer L.; Borland, Christina Z.; Stern, Michael J.

1998-01-01

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans. PMID:9618511

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter.

PubMed

Matsumoto, Yusuke; Ohta, Keisuke; Goto, Hideo; Nishio, Machiko

2016-07-01

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25-30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Distributed Representation of Visual Objects by Single Neurons in the Human Brain

PubMed Central

Valdez, André B.; Papesh, Megan H.; Treiman, David M.; Smith, Kris A.; Goldinger, Stephen D.

2015-01-01

It remains unclear how single neurons in the human brain represent whole-object visual stimuli. While recordings in both human and nonhuman primates have shown distributed representations of objects (many neurons encoding multiple objects), recordings of single neurons in the human medial temporal lobe, taken as subjects' discriminated objects during multiple presentations, have shown gnostic representations (single neurons encoding one object). Because some studies suggest that repeated viewing may enhance neural selectivity for objects, we had human subjects discriminate objects in a single, more naturalistic viewing session. We found that, across 432 well isolated neurons recorded in the hippocampus and amygdala, the average fraction of objects encoded was 26%. We also found that more neurons encoded several objects versus only one object in the hippocampus (28 vs 18%, p < 0.001) and in the amygdala (30 vs 19%, p < 0.001). Thus, during realistic viewing experiences, typical neurons in the human medial temporal lobe code for a considerable range of objects, across multiple semantic categories. PMID:25834044

Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.

PubMed

Lee, Soo Chan; Li, Alicia; Calo, Silvia; Heitman, Joseph

2013-01-01

Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO₂ conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.

Developmental programming of growth: genetic variant in GH2 gene encoding placental growth hormone contributes to adult height determination.

PubMed

Timasheva, Y; Putku, M; Kivi, R; Kožich, V; Männik, J; Laan, M

2013-11-01

Given the physiological role of placental growth hormone (PGH) during intrauterine development and growth, genetic variation in the coding Growth hormone 2 (GH2) gene may modulate developmental programming of adult stature. Two major GH2 variants were described worldwide, determined by single polymorphism (rs2006123; c.171 + 50C > A). We sought to study whether GH2 variants may contribute to adult anthropometric measurements. Genotyping of GH2 SNP rs2006123 by RFLP, testing its genetic association with adult height and Body Mass Index (BMI) by linear regression analysis, and combining the results of three individual study samples in meta-analysis. HYPEST (Estonia), n = 1464 (506 men/958 women), CADCZ (Czech), n = 871 (518/353); UFA (Bashkortostan), n = 954 (655/299); meta-analysis, n = 3289 (1679/1610). Meta-analysis across HYPEST, CADCZ and UFA samples (n = 3289) resulted in significant association of GH2 rs2006123 with height (recessive model: AA-homozygote effect: beta (SE) = 1.26 (0.46), P = 5.90 × 10⁻³; additive model: A-allele effect: beta (SE) = 0.45 (0.18), P = 1.40 × 10⁻²). Among men (n = 1679), the association of the A-allele with taller stature remained significant after multiple-testing correction (additive effect: beta = 0.86 (0.28), P = 1.83 × 10⁻³). No association was detected with BMI. Notably, rs2006123 was in strong LD (r² ≥ 0.87) with SNPs significantly associated with height (rs2665838, rs7209435, rs11658329) and mapped near GH2 in three independent meta-analyses of GWA studies. This is the first study demonstrating a link between a placental gene variant and programming of growth potential in adulthood. The detected association between PGH encoding GH2 and adult height promotes further research on the role of placental genes in prenatal programming of human metabolism. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Dissociations within human hippocampal subregions during encoding and retrieval of spatial information.

PubMed

Suthana, Nanthia; Ekstrom, Arne; Moshirvaziri, Saba; Knowlton, Barbara; Bookheimer, Susan

2011-07-01

Although the hippocampus is critical for the formation and retrieval of spatial memories, it is unclear how subregions are differentially involved in these processes. Previous high-resolution functional magnetic resonance imaging (fMRI) studies have shown that CA2, CA3, and dentate gyrus (CA23DG) regions support the encoding of novel associations, whereas the subicular cortices support the retrieval of these learned associations. Whether these subregions are used in humans during encoding and retrieval of spatial information has yet to be explored. Using high-resolution fMRI (1.6 mm × 1.6-mm in-plane), we found that activity within the right CA23DG increased during encoding compared to retrieval. Conversely, right subicular activity increased during retrieval compared to encoding of spatial associations. These results are consistent with the previous studies illustrating dissociations within human hippocampal subregions and further suggest that these regions are similarly involved during the encoding and retrieval of spatial information. Copyright © 2010 Wiley-Liss, Inc.

Effect of Unpleasant Loud Noise on Hippocampal Activities during Picture Encoding: An fMRI Study

ERIC Educational Resources Information Center

Hirano, Yoshiyuki; Fujita, Masafumi; Watanabe, Kazuko; Niwa, Masami; Takahashi, Toru; Kanematsu, Masayuki; Ido, Yasushi; Tomida, Mihoko; Onozuka, Minoru

2006-01-01

The functional link between the amygdala and hippocampus in humans has not been well documented. We examined the effect of unpleasant loud noise on hippocampal and amygdaloid activities during picture encoding by means of fMRI, and on the correct response in humans. The noise reduced activity in the hippocampus during picture encoding, decreased…

Growth factor transgenes interactively regulate articular chondrocytes.

PubMed

Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

2013-04-01

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. Copyright © 2012 Wiley Periodicals, Inc.

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Nevels, Michael; Dobner, Thomas

2007-01-01

The last 50 yr of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells and athymic nude mice.

Material and methods to increase plant growth and yield

DOEpatents

Kirst, Matias

2015-09-15

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Materials and methods to increase plant growth and yield

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

Kirst, Matias

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Gene transfer of a naked plasmid (pUDK-HGF) encoding human hepatocyte growth factor attenuates skin/muscle incision and retraction-induced chronic post-surgical pain in rats.

PubMed

Hu, C; Lu, Y; Chen, X; Wu, Z; Zhang, Q

2018-05-01

Chronic post-surgical pain (CPSP) remains a major clinical problem and is often refractory to current treatments. New analgesic medications and strategies for pain relief are needed. Hepatocyte growth factor (HGF) is known to be a multi-functional growth factor and regulates various biological activities. We investigated the analgesic effect and underlying mechanism of plasmid pUDK-HGF encoding human HGF gene on CPSP induced by skin/muscle incision and retraction (SMIR) in rats. The possible changes of inflammatory factors, glial cell activation and pain sensitivity after pUDK-HGF administration were investigated by ELISA, western blot and Von Frey tests, respectively. In behavioural assays, we found that a single intramuscular or intrathecal injection of pUDK-HGF significantly attenuated mechanical hypersensitivity to von Frey stimulation of plantar ipsilateral hind paw after SMIR. Intramuscular injection of pUDK-HGF promoted blood flow and proliferation of satellite cells and inhibited inflammatory cells recruitment, collagen accumulation and expression of pronociceptive factors. Intrathecal injection of pUDK-HGF inhibited activation of spinal glial cells and production of inflammatory mediators induced by SMIR. pUDK-HGF has a strong analgesic potency and efficacy in CPSP induced by SMIR in rats. This study highlights a new strategy for the treatment of CPSP. The CPSP occurs following various surgical procedures and remains a major clinical problem due to the lack of study on the mechanisms of CPSP. Our findings provide the first evidence that pUDK-HGF attenuates SMIR-induced pain behaviuors through peripheral or central mechanisms. The peripheral analgesic effect of pUDK-HGF is associated with promoting tissue repair and inhibiting inflammatory response; furthermore, pUDK-HGF inhibits activation of spinal glial cells and overexpression of inflammatory mediators in spinal cord. Therefore, naked pUDK-HGF may be a potential therapeutic strategy for treatment of CPSP in clinic. © 2018 European Pain Federation - EFIC®.

Anti-Tumor Effect of the Alphavirus-based Virus-like Particle Vector Expressing Prostate-Specific Antigen in a HLA-DR Transgenic Mouse Model of Prostate Cancer

PubMed Central

Riabov, V.; Tretyakova, I.; Alexander, R. B.; Pushko, P.; Klyushnenkova, E. N.

2015-01-01

The goal of this study was to determine if an alphavirus-based vaccine encoding human Prostate-Specific Antigen (PSA) could generate an effective anti-tumor immune response in a stringent mouse model of prostate cancer. DR2bxPSA F1 male mice expressing human PSA and HLA-DRB1*1501 transgenes were vaccinated with virus-like particle vector encoding PSA (VLPV-PSA) followed by the challenge with Transgenic Adenocarcinoma of Mouse Prostate cells engineered to express PSA (TRAMP-PSA). PSA-specific cellular and humoral immune responses were measured before and after tumor challenge. PSA and CD8 reactivity in the tumors was detected by immunohistochemistry. Tumor growth was compared in vaccinated and control groups. We found that VLPV-PSA could infect mouse dendritic cells in vitro and induce a robust PSA-specific immune response in vivo. A substantial proportion of splenic CD8+ T cells (19.6±7.4%) produced IFNγ in response to the immunodominant peptide PSA65–73. In the blood of vaccinated mice, 18.4±4.1% of CD8+ T cells were PSA-specific as determined by the staining with H-2Db/PSA65–73 dextramers. VLPV-PSA vaccination also strongly stimulated production of IgG2a/b anti-PSA antibodies. Tumors in vaccinated mice showed low levels of PSA expression and significant CD8 T cell infiltration. Tumor growth in VLPV-PSA vaccinated mice was significantly delayed at early time points (p=0.002, Gehan-Breslow test). Our data suggest that TC-83-based VLPV-PSA vaccine can efficiently overcome immune tolerance to PSA, mediate rapid clearance of PSA-expressing tumor cells and delay tumor growth. The VLPV-PSA vaccine will undergo further testing for the immunotherapy of prostate cancer. PMID:26319744

Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures.

PubMed Central

Gomes, S L; Gober, J W; Shapiro, L

1990-01-01

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions. Images PMID:2345134

Silver Nanoparticles: Two-Faced Neuronal Differentiation-Inducing Material in Neuroblastoma (SH-SY5Y) Cells.

PubMed

Abdal Dayem, Ahmed; Lee, Soo Bin; Choi, Hye Yeon; Cho, Ssang-Goo

2018-05-15

We have previously demonstrated the potential of biologically synthesized silver nanoparticles (AgNP) in the induction of neuronal differentiation of human neuroblastoma, SH-SY5Y cells; we aimed herein to unveil its molecular mechanism in comparison to the well-known neuronal differentiation-inducing agent, all-trans-retinoic acid (RA). AgNP-treated SH-SY5Y cells showed significantly higher reactive oxygen species (ROS) generation, stronger mitochondrial membrane depolarization, lower dual-specificity phosphatase expression, higher extracellular-signal-regulated kinase (ERK) phosphorylation, lower AKT phosphorylation, and lower expression of the genes encoding the antioxidant enzymes than RA-treated cells. Notably, pretreatment with N -acetyl-l-cysteine significantly abolished AgNP-induced neuronal differentiation, but not in that induced by RA. ERK inhibition, but not AKT inhibition, suppresses neurite growth that is induced by AgNP. Taken together, our results uncover the pivotal contribution of ROS in the AgNP-induced neuronal differentiation mechanism, which is different from that of RA. However, the negative consequence of AgNP-induced neurite growth may be high ROS generation and the downregulation of the expression of the genes encoding the antioxidant enzymes, which prompts the future consideration and an in-depth study of the application of AgNP-differentiated cells in neurodegenerative disease therapy.

Developing a hippocampal neural prosthetic to facilitate human memory encoding and recall.

PubMed

Hampson, Robert E; Song, Dong; Robinson, Brian S; Fetterhoff, Dustin; Dakos, Alexander S; Roeder, Brent M; She, Xiwei; Wicks, Robert T; Witcher, Mark R; Couture, Daniel E; Laxton, Adrian W; Munger-Clary, Heidi; Popli, Gautam; Sollman, Myriam J; Whitlow, Christopher T; Marmarelis, Vasilis Z; Berger, Theodore W; Deadwyler, Sam A

2018-06-01

We demonstrate here the first successful implementation in humans of a proof-of-concept system for restoring and improving memory function via facilitation of memory encoding using the patient's own hippocampal spatiotemporal neural codes for memory. Memory in humans is subject to disruption by drugs, disease and brain injury, yet previous attempts to restore or rescue memory function in humans typically involved only nonspecific, modulation of brain areas and neural systems related to memory retrieval. We have constructed a model of processes by which the hippocampus encodes memory items via spatiotemporal firing of neural ensembles that underlie the successful encoding of short-term memory. A nonlinear multi-input, multi-output (MIMO) model of hippocampal CA3 and CA1 neural firing is computed that predicts activation patterns of CA1 neurons during the encoding (sample) phase of a delayed match-to-sample (DMS) human short-term memory task. MIMO model-derived electrical stimulation delivered to the same CA1 locations during the sample phase of DMS trials facilitated short-term/working memory by 37% during the task. Longer term memory retention was also tested in the same human subjects with a delayed recognition (DR) task that utilized images from the DMS task, along with images that were not from the task. Across the subjects, the stimulated trials exhibited significant improvement (35%) in both short-term and long-term retention of visual information. These results demonstrate the facilitation of memory encoding which is an important feature for the construction of an implantable neural prosthetic to improve human memory.

Developing a hippocampal neural prosthetic to facilitate human memory encoding and recall

NASA Astrophysics Data System (ADS)

Hampson, Robert E.; Song, Dong; Robinson, Brian S.; Fetterhoff, Dustin; Dakos, Alexander S.; Roeder, Brent M.; She, Xiwei; Wicks, Robert T.; Witcher, Mark R.; Couture, Daniel E.; Laxton, Adrian W.; Munger-Clary, Heidi; Popli, Gautam; Sollman, Myriam J.; Whitlow, Christopher T.; Marmarelis, Vasilis Z.; Berger, Theodore W.; Deadwyler, Sam A.

2018-06-01

Objective. We demonstrate here the first successful implementation in humans of a proof-of-concept system for restoring and improving memory function via facilitation of memory encoding using the patient’s own hippocampal spatiotemporal neural codes for memory. Memory in humans is subject to disruption by drugs, disease and brain injury, yet previous attempts to restore or rescue memory function in humans typically involved only nonspecific, modulation of brain areas and neural systems related to memory retrieval. Approach. We have constructed a model of processes by which the hippocampus encodes memory items via spatiotemporal firing of neural ensembles that underlie the successful encoding of short-term memory. A nonlinear multi-input, multi-output (MIMO) model of hippocampal CA3 and CA1 neural firing is computed that predicts activation patterns of CA1 neurons during the encoding (sample) phase of a delayed match-to-sample (DMS) human short-term memory task. Main results. MIMO model-derived electrical stimulation delivered to the same CA1 locations during the sample phase of DMS trials facilitated short-term/working memory by 37% during the task. Longer term memory retention was also tested in the same human subjects with a delayed recognition (DR) task that utilized images from the DMS task, along with images that were not from the task. Across the subjects, the stimulated trials exhibited significant improvement (35%) in both short-term and long-term retention of visual information. Significance. These results demonstrate the facilitation of memory encoding which is an important feature for the construction of an implantable neural prosthetic to improve human memory.

Rapid formation and flexible expression of memories of subliminal word pairs.

PubMed

Reber, Thomas P; Henke, Katharina

2011-01-01

Our daily experiences are incidentally and rapidly encoded as episodic memories. Episodic memories consist of numerous associations (e.g., who gave what to whom where and when) that can be expressed flexibly in new situations. Key features of episodic memory are speed of encoding, its associative nature, and its representational flexibility. Another defining feature of human episodic memory has been consciousness of encoding/retrieval. Here, we show that humans can rapidly form associations between subliminal words and minutes later retrieve these associations even if retrieval words were conceptually related to, but different from encoding words. Because encoding words were presented subliminally, associative encoding, and retrieval were unconscious. Unconscious association formation and retrieval were dependent on a preceding understanding of task principles. We conclude that key computations underlying episodic memory - rapid encoding and flexible expression of associations - can operate outside consciousness.

Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

PubMed

McBrayer, Samuel K; Olenchock, Benjamin A; DiNatale, Gabriel J; Shi, Diana D; Khanal, Januka; Jennings, Rebecca B; Novak, Jesse S; Oser, Matthew G; Robbins, Alissa K; Modiste, Rebecca; Bonal, Dennis; Moslehi, Javid; Bronson, Roderick T; Neuberg, Donna; Nguyen, Quang-De; Signoretti, Sabina; Losman, Julie-Aurore; Kaelin, William G

2018-04-17

Inactivation of the retinoblastoma gene ( RB1 ) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1 +/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1 +/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1 -null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Diversification of the insulin-like growth factor 1 gene in mammals.

PubMed

Rotwein, Peter

2017-01-01

Insulin-like growth factor 1 (IGF1), a small, secreted peptide growth factor, is involved in a variety of physiological and patho-physiological processes, including somatic growth, tissue repair, and metabolism of carbohydrates, proteins, and lipids. IGF1 gene expression appears to be controlled by several different signaling cascades in the few species in which it has been evaluated, with growth hormone playing a major role by activating a pathway involving the Stat5b transcription factor. Here, genes encoding IGF1 have been evaluated in 25 different mammalian species representing 15 different orders and ranging over ~180 million years of evolutionary diversification. Parts of the IGF1 gene have been fairly well conserved. Like rat Igf1 and human IGF1, 21 of 23 other genes are composed of 6 exons and 5 introns, and all 23 also contain recognizable tandem promoters, each with a unique leader exon. Exon and intron lengths are similar in most species, and DNA sequence conservation is moderately high in orthologous exons and proximal promoter regions. In contrast, putative growth hormone-activated Stat5b-binding enhancers found in analogous locations in rodent Igf1 and in human IGF1 loci, have undergone substantial variation in other mammals, and a processed retro-transposed IGF1 pseudogene is found in the sloth locus, but not in other mammalian genomes. Taken together, the fairly high level of organizational and nucleotide sequence similarity in the IGF1 gene among these 25 species supports the contention that some common regulatory pathways had existed prior to the beginning of mammalian speciation.

Determining the Neural Substrate for Encoding a Memory of Human Pain and the Influence of Anxiety

PubMed Central

Kong, Yazhuo; Tracey, Irene

2017-01-01

To convert a painful stimulus into a briefly maintainable construct when the painful stimulus is no longer accessible is essential to guide human behavior and avoid dangerous situations. Because of the aversive nature of pain, this encoding process might be influenced by emotional aspects and could thus vary across individuals, but we have yet to understand both the basic underlying neural mechanisms as well as potential interindividual differences. Using fMRI in combination with a delayed-discrimination task in healthy volunteers of both sexes, we discovered that brain regions involved in this working memory encoding process were dissociable according to whether the to-be-remembered stimulus was painful or not, with the medial thalamus and the rostral anterior cingulate cortex encoding painful and the primary somatosensory cortex encoding nonpainful stimuli. Encoding of painful stimuli furthermore significantly enhanced functional connectivity between the thalamus and medial prefrontal cortex (mPFC). With regards to emotional aspects influencing encoding processes, we observed that more anxious participants showed significant performance advantages when encoding painful stimuli. Importantly, only during the encoding of pain, the interindividual differences in anxiety were associated with the strength of coupling between medial thalamus and mPFC, which was furthermore related to activity in the amygdala. These results indicate not only that there is a distinct signature for the encoding of a painful experience in humans, but also that this encoding process involves a strong affective component. SIGNIFICANCE STATEMENT To convert the sensation of pain into a briefly maintainable construct is essential to guide human behavior and avoid dangerous situations. Although this working memory encoding process is implicitly contained in the majority of studies, the underlying neural mechanisms remain unclear. Using fMRI in a delayed-discrimination task, we found that the encoding of pain engaged the activation of the medial thalamus and the functional connectivity between the thalamus and medial prefrontal cortex. These fMRI data were directly and indirectly related to participants' self-reported trait and state anxiety. Our findings indicate that the mechanisms responsible for the encoding of noxious stimuli differ from those for the encoding of innocuous stimuli, and that these mechanisms are shaped by an individual's anxiety levels. PMID:29097595

Determining the Neural Substrate for Encoding a Memory of Human Pain and the Influence of Anxiety.

PubMed

Tseng, Ming-Tsung; Kong, Yazhuo; Eippert, Falk; Tracey, Irene

2017-12-06

To convert a painful stimulus into a briefly maintainable construct when the painful stimulus is no longer accessible is essential to guide human behavior and avoid dangerous situations. Because of the aversive nature of pain, this encoding process might be influenced by emotional aspects and could thus vary across individuals, but we have yet to understand both the basic underlying neural mechanisms as well as potential interindividual differences. Using fMRI in combination with a delayed-discrimination task in healthy volunteers of both sexes, we discovered that brain regions involved in this working memory encoding process were dissociable according to whether the to-be-remembered stimulus was painful or not, with the medial thalamus and the rostral anterior cingulate cortex encoding painful and the primary somatosensory cortex encoding nonpainful stimuli. Encoding of painful stimuli furthermore significantly enhanced functional connectivity between the thalamus and medial prefrontal cortex (mPFC). With regards to emotional aspects influencing encoding processes, we observed that more anxious participants showed significant performance advantages when encoding painful stimuli. Importantly, only during the encoding of pain, the interindividual differences in anxiety were associated with the strength of coupling between medial thalamus and mPFC, which was furthermore related to activity in the amygdala. These results indicate not only that there is a distinct signature for the encoding of a painful experience in humans, but also that this encoding process involves a strong affective component. SIGNIFICANCE STATEMENT To convert the sensation of pain into a briefly maintainable construct is essential to guide human behavior and avoid dangerous situations. Although this working memory encoding process is implicitly contained in the majority of studies, the underlying neural mechanisms remain unclear. Using fMRI in a delayed-discrimination task, we found that the encoding of pain engaged the activation of the medial thalamus and the functional connectivity between the thalamus and medial prefrontal cortex. These fMRI data were directly and indirectly related to participants' self-reported trait and state anxiety. Our findings indicate that the mechanisms responsible for the encoding of noxious stimuli differ from those for the encoding of innocuous stimuli, and that these mechanisms are shaped by an individual's anxiety levels. Copyright © 2017 Tseng et al.

Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

PubMed

Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

2016-06-01

Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter.

Face Encoding and Recognition in the Human Brain

NASA Astrophysics Data System (ADS)

Haxby, James V.; Ungerleider, Leslie G.; Horwitz, Barry; Maisog, Jose Ma.; Rapoport, Stanley I.; Grady, Cheryl L.

1996-01-01

A dissociation between human neural systems that participate in the encoding and later recognition of new memories for faces was demonstrated by measuring memory task-related changes in regional cerebral blood flow with positron emission tomography. There was almost no overlap between the brain structures associated with these memory functions. A region in the right hippocampus and adjacent cortex was activated during memory encoding but not during recognition. The most striking finding in neocortex was the lateralization of prefrontal participation. Encoding activated left prefrontal cortex, whereas recognition activated right prefrontal cortex. These results indicate that the hippocampus and adjacent cortex participate in memory function primarily at the time of new memory encoding. Moreover, face recognition is not mediated simply by recapitulation of operations performed at the time of encoding but, rather, involves anatomically dissociable operations.

Encoding of physics concepts: concreteness and presentation modality reflected by human brain dynamics.

PubMed

Lai, Kevin; She, Hsiao-Ching; Chen, Sheng-Chang; Chou, Wen-Chi; Huang, Li-Yu; Jung, Tzyy-Ping; Gramann, Klaus

2012-01-01

Previous research into working memory has focused on activations in different brain areas accompanying either different presentation modalities (verbal vs. non-verbal) or concreteness (abstract vs. concrete) of non-science concepts. Less research has been conducted investigating how scientific concepts are learned and further processed in working memory. To bridge this gap, the present study investigated human brain dynamics associated with encoding of physics concepts, taking both presentation modality and concreteness into account. Results of this study revealed greater theta and low-beta synchronization in the anterior cingulate cortex (ACC) during encoding of concrete pictures as compared to the encoding of both high and low imageable words. In visual brain areas, greater theta activity accompanying stimulus onsets was observed for words as compared to pictures while stronger alpha suppression was observed in responses to pictures as compared to words. In general, the EEG oscillation patterns for encoding words of different levels of abstractness were comparable but differed significantly from encoding of pictures. These results provide insights into the effects of modality of presentation on human encoding of scientific concepts and thus might help in developing new ways to better teach scientific concepts in class.

CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

PubMed

Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

2018-04-03

The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells

PubMed Central

Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

2018-01-01

The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination. PMID:29707137

Rapid Formation and Flexible Expression of Memories of Subliminal Word Pairs

PubMed Central

Reber, Thomas P.; Henke, Katharina

2011-01-01

Our daily experiences are incidentally and rapidly encoded as episodic memories. Episodic memories consist of numerous associations (e.g., who gave what to whom where and when) that can be expressed flexibly in new situations. Key features of episodic memory are speed of encoding, its associative nature, and its representational flexibility. Another defining feature of human episodic memory has been consciousness of encoding/retrieval. Here, we show that humans can rapidly form associations between subliminal words and minutes later retrieve these associations even if retrieval words were conceptually related to, but different from encoding words. Because encoding words were presented subliminally, associative encoding, and retrieval were unconscious. Unconscious association formation and retrieval were dependent on a preceding understanding of task principles. We conclude that key computations underlying episodic memory – rapid encoding and flexible expression of associations – can operate outside consciousness. PMID:22125545

Paracrine-Mediated Neuroprotection and Neuritogenesis of Axotomised Retinal Ganglion Cells by Human Dental Pulp Stem Cells: Comparison with Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

PubMed Central

Mead, Ben; Logan, Ann; Berry, Martin

2014-01-01

We have investigated and compared the neurotrophic activity of human dental pulp stem cells (hDPSC), human bone marrow-derived mesenchymal stem cells (hBMSC) and human adipose-derived stem cells (hAMSC) on axotomised adult rat retinal ganglion cells (RGC) in vitro in order to evaluate their therapeutic potential for neurodegenerative conditions of RGC. Using the transwell system, RGC survival and length/number of neurites were quantified in coculture with stem cells in the presence or absence of specific Fc-receptor inhibitors to determine the role of NGF, BDNF, NT-3, VEGF, GDNF, PDGF-AA and PDGF-AB/BB in stem cell-mediated RGC neuroprotection and neuritogenesis. Conditioned media, collected from cultured hDPSC/hBMSC/hAMSC, were assayed for the secreted growth factors detailed above using ELISA. PCR array determined the hDPSC, hBMSC and hAMSC expression of genes encoding 84 growth factors and receptors. The results demonstrated that hDPSC promoted significantly more neuroprotection and neuritogenesis of axotomised RGC than either hBMSC or hAMSC, an effect that was neutralized after the addition of specific Fc-receptor inhibitors. hDPSC secreted greater levels of various growth factors including NGF, BDNF and VEGF compared with hBMSC/hAMSC. The PCR array confirmed these findings and identified VGF as a novel potentially therapeutic hDPSC-derived neurotrophic factor (NTF) with significant RGC neuroprotective properties after coculture with axotomised RGC. In conclusion, hDPSC promoted significant multi-factorial paracrine-mediated RGC survival and neurite outgrowth and may be considered a potent and advantageous cell therapy for retinal nerve repair. PMID:25290916

p53 functions as a cell cycle control protein in osteosarcomas.

PubMed Central

Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-01-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Images PMID:2233717

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

PubMed Central

Lee, I. Russel; Chow, Eve W. L.; Morrow, Carl A.; Djordjevic, Julianne T.; Fraser, James A.

2011-01-01

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine—three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°–40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection. PMID:21441208

A Critical Role of Zinc Importer AdcABC in Group A Streptococcus-Host Interactions During Infection and Its Implications for Vaccine Development.

PubMed

Makthal, Nishanth; Nguyen, Kimberly; Do, Hackwon; Gavagan, Maire; Chandrangsu, Pete; Helmann, John D; Olsen, Randall J; Kumaraswami, Muthiah

2017-07-01

Bacterial pathogens must overcome host immune mechanisms to acquire micronutrients for successful replication and infection. Streptococcus pyogenes, also known as group A streptococcus (GAS), is a human pathogen that causes a variety of clinical manifestations, and disease prevention is hampered by lack of a human GAS vaccine. Herein, we report that the mammalian host recruits calprotectin (CP) to GAS infection sites and retards bacterial growth by zinc limitation. However, a GAS-encoded zinc importer and a nuanced zinc sensor aid bacterial defense against CP-mediated growth inhibition and contribute to GAS virulence. Immunization of mice with the extracellular component of the zinc importer confers protection against systemic GAS challenge. Together, we identified a key early stage host-GAS interaction and translated that knowledge into a novel vaccine strategy against GAS infection. Furthermore, we provided evidence that a similar struggle for zinc may occur during other streptococcal infections, which raises the possibility of a broad-spectrum prophylactic strategy against multiple streptococcal pathogens. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Targeting Gas6/TAM in cancer cells and tumor microenvironment.

PubMed

Wu, Guiling; Ma, Zhiqiang; Cheng, Yicheng; Hu, Wei; Deng, Chao; Jiang, Shuai; Li, Tian; Chen, Fulin; Yang, Yang

2018-01-31

Growth arrest-specific 6, also known as Gas6, is a human gene encoding the Gas6 protein, which was originally found to be upregulated in growth-arrested fibroblasts. Gas6 is a member of the vitamin K-dependent family of proteins expressed in many human tissues and regulates several biological processes in cells, including proliferation, survival and migration, by binding to its receptors Tyro3, Axl and Mer (TAM). In recent years, the roles of Gas6/TAM signalling in cancer cells and the tumour microenvironment have been studied, and some progress has made in targeted therapy, providing new potential directions for future investigations of cancer treatment. In this review, we introduce the Gas6 and TAM receptors and describe their involvement in different cancers and discuss the roles of Gas6 in cancer cells, the tumour microenvironment and metastasis. Finally, we introduce recent studies on Gas6/TAM targeting in cancer therapy, which will assist in the experimental design of future analyses and increase the potential use of Gas6 as a therapeutic target for cancer.

The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.

PubMed

Lin, Wei-Jye; Salton, Stephen R

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

The Regulated Secretory Pathway and Human Disease: Insights from Gene Variants and Single Nucleotide Polymorphisms

PubMed Central

Lin, Wei-Jye; Salton, Stephen R.

2013-01-01

The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired. PMID:23964269

TCF and Groucho-related genes influence pituitary growth and development.

PubMed

Brinkmeier, Michelle L; Potok, Mary Anne; Cha, Kelly B; Gridley, Thomas; Stifani, Stefano; Meeldijk, Jan; Clevers, Hans; Camper, Sally A

2003-11-01

Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.

Distributed representation of visual objects by single neurons in the human brain.

PubMed

Valdez, André B; Papesh, Megan H; Treiman, David M; Smith, Kris A; Goldinger, Stephen D; Steinmetz, Peter N

2015-04-01

It remains unclear how single neurons in the human brain represent whole-object visual stimuli. While recordings in both human and nonhuman primates have shown distributed representations of objects (many neurons encoding multiple objects), recordings of single neurons in the human medial temporal lobe, taken as subjects' discriminated objects during multiple presentations, have shown gnostic representations (single neurons encoding one object). Because some studies suggest that repeated viewing may enhance neural selectivity for objects, we had human subjects discriminate objects in a single, more naturalistic viewing session. We found that, across 432 well isolated neurons recorded in the hippocampus and amygdala, the average fraction of objects encoded was 26%. We also found that more neurons encoded several objects versus only one object in the hippocampus (28 vs 18%, p < 0.001) and in the amygdala (30 vs 19%, p < 0.001). Thus, during realistic viewing experiences, typical neurons in the human medial temporal lobe code for a considerable range of objects, across multiple semantic categories. Copyright © 2015 the authors 0270-6474/15/355180-07$15.00/0.

Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity.

PubMed

Hwang, Kyung-Sun; Cho, Won-Kyung; Yoo, Jinsang; Seong, Young Rim; Kim, Bum-Kyeng; Kim, Samyong; Im, Dong-Soo

2004-06-01

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5'-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32-), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32- produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32- compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32- contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18. Copyright 2004 Nature Publishing Group

The hippocampal formation participates in novel picture encoding: evidence from functional magnetic resonance imaging.

PubMed Central

Stern, C E; Corkin, S; González, R G; Guimaraes, A R; Baker, J R; Jennings, P J; Carr, C A; Sugiura, R M; Vedantham, V; Rosen, B R

1996-01-01

Considerable evidence exists to support the hypothesis that the hippocampus and related medial temporal lobe structures are crucial for the encoding and storage of information in long-term memory. Few human imaging studies, however, have successfully shown signal intensity changes in these areas during encoding or retrieval. Using functional magnetic resonance imaging (fMRI), we studied normal human subjects while they performed a novel picture encoding task. High-speed echo-planar imaging techniques evaluated fMRI signal changes throughout the brain. During the encoding of novel pictures, statistically significant increases in fMRI signal were observed bilaterally in the posterior hippocampal formation and parahippocampal gyrus and in the lingual and fusiform gyri. To our knowledge, this experiment is the first fMRI study to show robust signal changes in the human hippocampal region. It also provides evidence that the encoding of novel, complex pictures depends upon an interaction between ventral cortical regions, specialized for object vision, and the hippocampal formation and parahippocampal gyrus, specialized for long-term memory. Images Fig. 1 Fig. 3 PMID:8710927

Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity of a human breast cancer cell line in vitro and in vivo.

PubMed

Spiridon, Camelia I; Ghetie, Maria-Ana; Uhr, Jonathan; Marches, Radu; Li, Jia-Ling; Shen, Guo-Liang; Vitetta, Ellen S

2002-06-01

Her-2 (p185(erbB-2)) is a transmembrane tyrosine kinase receptor, which is encoded by the Her-2/neu proto-oncogene. Her-2 is overexpressed on 30% of highly malignant breast cancers. Monoclonal antibodies (MAbs) against Her-2 inhibit the growth of Her-2-overexpressing tumor cells and this occurs by a variety of mechanisms. One such MAb, Herceptin (Trastuzumab), has been approved for human use. We have generated a panel of murine anti-Her-2 MAbs against nine different epitopes on the extracellular domain of Her-2 and have evaluated the antitumor activity of three of these MAbs alone and in combination, both in vitro and in vivo. We found that MAbs (against different epitopes) make a highly effective mixture, which was more effective than the individual MAbs in treating s.c. tumor nodules of BT474 cells in SCID mice. In vitro, the MAb mixture was also more effective than the single MAbs in inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, inhibiting cell growth and inducing apoptosis, and inhibiting the secretion of vascular endothelial growth factor. Taken together, these activities might explain the superior performance of the MAb mixture in vivo.

Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer

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

Yoshida, Michihiro C.; Wada, Makio; Satoh, Hitoshi

1988-07-01

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. The authors have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of amore » panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.« less

Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

PubMed Central

Jia, Z P; McCullough, N; Martel, R; Hemmingsen, S; Young, P G

1992-01-01

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance. Images PMID:1314171

The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence.

PubMed

Hissen, Anna H T; Wan, Adrian N C; Warwas, Mark L; Pinto, Linda J; Moore, Margo M

2005-09-01

Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes l-ornithine N(5)-oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal l-ornithine N(5)-oxygenases. A stable DeltasidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N',N",N'''-triacetylfusarinine C (TAF) and ferricrocin. Growth of the DeltasidA strain was the same as that of the wild type in rich media; however, the DeltasidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the DeltasidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the DeltasidA strain was unable to remove iron from human transferrin. A rescued strain (DeltasidA + sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the DeltasidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

Role for transforming growth factor-beta1 in alport renal disease progression.

PubMed

Sayers, R; Kalluri, R; Rodgers, K D; Shield, C F; Meehan, D T; Cosgrove, D

1999-11-01

Alport syndrome results from mutations in either the alpha3(IV), alpha4(IV), or alpha5(IV) collagen genes. The disease is characterized by a progressive glomerulonephritis usually associated with a high-frequency sensorineural hearing loss. A mouse model for an autosomal form of Alport syndrome [collagen alpha3(IV) knockout] was produced and characterized. In this study, the model was exploited to demonstrate a potential role for transforming growth factor-beta1 (TGF-beta1) in Alport renal disease pathogenesis. Kidneys from normal and Alport mice, taken at different stages during the course of renal disease progression, were analyzed by Northern blot, in situ hybridization, and immunohistology for expression of TGF-beta1 and components of the extracellular matrix. Normal and Alport human kidney was examined for TGF-beta1 expression using RNase protection. The mRNAs encoding TGF-beta1 (in both mouse and human), entactin, fibronectin, and the collagen alpha1(IV) and alpha2(IV) chains were significantly induced in total kidney as a function of Alport renal disease progression. The induction of these specific mRNAs was observed in the glomerular podocytes of animals with advanced disease. Type IV collagen, laminin-1, and fibronectin were markedly elevated in the tubulointerstitium at 10 weeks, but not at 6 weeks, suggesting that elevated expression of specific mRNAs on Northern blots reflects events associated with tubulointerstitial fibrosis. The concomitant accumulation of mRNAs encoding TGF-beta1 and extracellular matrix components in the podocytes of diseased kidneys may reflect key events in Alport renal disease progression. These data suggest a role for TGF-beta1 in both glomerular and tubulointerstitial damage associated with Alport syndrome.

Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals.

PubMed

Theilmann, Mia C; Goh, Yong Jun; Nielsen, Kristian Fog; Klaenhammer, Todd R; Barrangou, Rodolphe; Abou Hachem, Maher

2017-11-21

Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host. IMPORTANCE Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by Lactobacillus acidophilus and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota. Copyright © 2017 Theilmann et al.

Draft Map of Human Proteome Published | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a recently published article in the journal Nature, researchers have developed a draft map of the human proteome.  Striving for the protein equivalent of the Human Genome Project, an international team of researchers has created an initial catalog of the human proteome. In total, using 30 different human tissues, the researchers identified proteins encoded by 17,294 genes, which is approximately 84 percent of all of the genes in the human genome predicted to encode proteins.

[The ENCODE project and functional genomics studies].

PubMed

Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

2014-03-01

Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

Relative cue encoding in the context of sophisticated models of categorization: Separating information from categorization.

PubMed

Apfelbaum, Keith S; McMurray, Bob

2015-08-01

Traditional studies of human categorization often treat the processes of encoding features and cues as peripheral to the question of how stimuli are categorized. However, in domains where the features and cues are less transparent, how information is encoded prior to categorization may constrain our understanding of the architecture of categorization. This is particularly true in speech perception, where acoustic cues to phonological categories are ambiguous and influenced by multiple factors. Here, it is crucial to consider the joint contributions of the information in the input and the categorization architecture. We contrasted accounts that argue for raw acoustic information encoding with accounts that posit that cues are encoded relative to expectations, and investigated how two categorization architectures-exemplar models and back-propagation parallel distributed processing models-deal with each kind of information. Relative encoding, akin to predictive coding, is a form of noise reduction, so it can be expected to improve model accuracy; however, like predictive coding, the use of relative encoding in speech perception by humans is controversial, so results are compared to patterns of human performance, rather than on the basis of overall accuracy. We found that, for both classes of models, in the vast majority of parameter settings, relative cues greatly helped the models approximate human performance. This suggests that expectation-relative processing is a crucial precursor step in phoneme categorization, and that understanding the information content is essential to understanding categorization processes.

Relative cue encoding in the context of sophisticated models of categorization: Separating information from categorization

PubMed Central

McMurray, Bob

2014-01-01

Traditional studies of human categorization often treat the processes of encoding features and cues as peripheral to the question of how stimuli are categorized. However, in domains where the features and cues are less transparent, how information is encoded prior to categorization may constrain our understanding of the architecture of categorization. This is particularly true in speech perception, where acoustic cues to phonological categories are ambiguous and influenced by multiple factors. Here, it is crucial to consider the joint contributions of the information in the input and the categorization architecture. We contrasted accounts that argue for raw acoustic information encoding with accounts that posit that cues are encoded relative to expectations, and investigated how two categorization architectures—exemplar models and back-propagation parallel distributed processing models—deal with each kind of information. Relative encoding, akin to predictive coding, is a form of noise reduction, so it can be expected to improve model accuracy; however, like predictive coding, the use of relative encoding in speech perception by humans is controversial, so results are compared to patterns of human performance, rather than on the basis of overall accuracy. We found that, for both classes of models, in the vast majority of parameter settings, relative cues greatly helped the models approximate human performance. This suggests that expectation-relative processing is a crucial precursor step in phoneme categorization, and that understanding the information content is essential to understanding categorization processes. PMID:25475048

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

PubMed Central

Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

2013-01-01

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the ‘normal’ small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells. PMID:24345856

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes

PubMed Central

Khuu, Cuong; Jevnaker, Anne-Marthe; Bryne, Magne; Osmundsen, Harald

2014-01-01

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40–50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92—or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed—about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to “Cellular Growth and Proliferation” and “Cell Cycle.” Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants. PMID:25202322

Encoder: A Connectionist Model of How Learning to Visually Encode Fixated Text Images Improves Reading Fluency

ERIC Educational Resources Information Center

Martin, Gale L.

2004-01-01

This article proposes that visual encoding learning improves reading fluency by widening the span over which letters are recognized from a fixated text image so that fewer fixations are needed to cover a text line. Encoder is a connectionist model that learns to convert images like the fixated text images human readers encode into the…

The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

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

Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst, E-mail: e.wolvetang@uq.edu.au

2012-05-15

The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatmentsmore » we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.« less

Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58.

PubMed

Mitsui, Ryoji; Katayama, Hiroko; Tanaka, Mitsuo

2015-07-01

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

2016-03-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

PubMed

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

PubMed Central

2014-01-01

Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

PubMed Central

Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E.; Posey, Avery D.; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C.; June, Carl H.

2015-01-01

This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet, EOMES and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-kB, Akt, Erk and NFAT. The propagated CAR T cells retained a diverse TCR repertoire and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore the design of CARs that have a non-constitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or non-constitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

PubMed

Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

2015-04-01

This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. ©2015 American Association for Cancer Research.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p.

PubMed

Cherkasova, Vera; Maury, Luis Lopez; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J

2012-02-01

Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p

PubMed Central

Cherkasova, Vera; Lopez Maury, Luis; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J.

2012-01-01

Deletion of the sla1+ gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1+ have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1–like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1+ (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1+ regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae. PMID:22160596

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius

DOE PAGES

Lee, Brady D.; Apel, William A.; DeVeaux, Linda C.; ...

2017-08-03

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in this paper in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grewmore » while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Finally, microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.« less

Methods for transforming and expression screening of filamentous fungal cells with a DNA library

DOEpatents

Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

2015-06-02

The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius

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

Lee, Brady D.; Apel, William A.; DeVeaux, Linda C.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in this paper in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grewmore » while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Finally, microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.« less

Transgenic plants with increased calcium stores

NASA Technical Reports Server (NTRS)

Robertson, Dominique (Inventor); Wyatt, Sarah (Inventor); Tsou, Pei-Lan (Inventor); Boss, Wendy (Inventor)

2004-01-01

The present invention provides transgenic plants over-expressing a transgene encoding a calcium-binding protein or peptide (CaBP). Preferably, the CaBP is a calcium storage protein and over-expression thereof does not have undue adverse effects on calcium homeostasis or biochemical pathways that are regulated by calcium. In preferred embodiments, the CaBP is calreticulin (CRT) or calsequestrin. In more preferred embodiments, the CaBP is the C-domain of CRT, a fragment of the C-domain, or multimers of the foregoing. In other preferred embodiments, the CaBP is localized to the endoplasmic reticulum by operatively associating the transgene encoding the CaBP with an endoplasmic reticulum localization peptide. Alternatively, the CaBP is targeted to any other sub-cellular compartment that permits the calcium to be stored in a form that is biologically available to the plant. Also provided are methods of producing plants with desirable phenotypic traits by transformation of the plant with a transgene encoding a CaBP. Such phenotypic traits include increased calcium storage, enhanced resistance to calcium-limiting conditions, enhanced growth and viability, increased disease and stress resistance, enhanced flower and fruit production, reduced senescence, and a decreased need for fertilizer production. Further provided are plants with enhanced nutritional value as human food or animal feed.

Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus.

PubMed

Brautaset, Trygve; Jakobsen M, Øyvind M; Flickinger, Michael C; Valla, Svein; Ellingsen, Trond E

2004-03-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

PubMed Central

Brautaset, Trygve; Jakobsen, Øyvind M.; Flickinger, Michael C.; Valla, Svein; Ellingsen, Trond E.

2004-01-01

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs. PMID:14973041

Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

PubMed

Han, Gil-Soo; Carman, George M

2017-08-11

The PAH1 -encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae , exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1 -encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1 Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1 Δ mutant is induced through the inositol-sensitive upstream activation sequence (UAS INO ), a cis -acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UAS INO mutation suppressed pah1 Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1 -encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1 Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

NASA Astrophysics Data System (ADS)

Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.

2002-05-01

The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.

Frequent Infection of Human Cancer Xenografts with Murine Endogenous Retroviruses in Vivo

PubMed Central

Naseer, Asif; Terry, Anne; Gilroy, Kathryn; Kilbey, Anna; Watts, Ciorsdaidh; Mackay, Nancy; Bell, Margaret; Mason, Susan; Blyth, Karen; Cameron, Ewan; Neil, James C.

2015-01-01

Infection of human cancer xenografts in mice with murine leukemia viruses (MLVs) is a long-standing observation, but the likelihood of infection in vivo and its biological consequences are poorly understood. We therefore conducted a prospective study in commonly used xenograft recipient strains. From BALB/c nude mice engrafted with MCF7 human mammary carcinoma cells, we isolated a virus that was virtually identical to Bxv1, a locus encoding replication-competent xenotropic MLV (XMLV). XMLV was detected in 9/17 (53%) independently isolated explants. XMLV was not found in primary leukemias or in THP1 leukemia cells grown in Bxv1-negative NSG (NOD/SCID/γCnull) mice, although MCF7 explants harbored replication-defective MLV proviruses. To assess the significance of infection for xenograft behavior in vivo, we examined changes in growth and global transcription in MCF7 and the highly susceptible Raji Burkitt lymphoma cell line chronically infected with XMLV. Raji cells showed a stronger transcriptional response that included up-regulation of chemokines and effectors of innate antiviral immunity. In conclusion, the risk of de novo XMLV infection of xenografts is high in Bxv1 positive mice, while infection can have positive or negative effects on xenograft growth potential with significant consequences for interpretation of many xenograft studies. PMID:25912714

Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis.

PubMed

López-Martín, José M; Salviati, Leonardo; Trevisson, Eva; Montini, Giovanni; DiMauro, Salvatore; Quinzii, Catarina; Hirano, Michio; Rodriguez-Hernandez, Angeles; Cordero, Mario D; Sánchez-Alcázar, José A; Santos-Ocaña, Carlos; Navas, Plácido

2007-05-01

Coenzyme Q(10) (CoQ(10)) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ(10) supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ(10) deficiency, a homozygous mutation was identified in the CoQ(10) biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ(6) concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33-45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ(10) supplementation, and growth rate was restored in these cells by either CoQ(10) or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ(10) deficiency. Moreover, we demonstrate that CoQ(10) deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.

MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

PubMed

Vecchione, A; Fassan, M; Anesti, V; Morrione, A; Goldoni, S; Baldassarre, G; Byrne, D; D'Arca, D; Palazzo, J P; Lloyd, J; Scorrano, L; Gomella, L G; Iozzo, R V; Baffa, R

2009-01-15

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

An anti-VEGF ribozyme embedded within the adenoviral VAI sequence inhibits glioblastoma cell angiogenic potential in vitro.

PubMed

Ciafrè, Silvia Anna; Niola, Francesco; Wannenes, Francesca; Farace, Maria Giulia

2004-01-01

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis, where it functions as one of the major angiogenic factors sustaining growth and draining catabolites. In this study, we developed an anti-VEGF ribozyme targeted to the 5' part of human VEGF mRNA. We endowed this ribozyme with an additional feature expected to improve its activity in vivo, by cloning it into a VAI transcriptional cassette. VAI is originally part of the adenovirus genome, and is characterized by high transcription rates, good stability due to its strong secondary structure and cytoplasmic localization. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secreted in the extracellular medium, indicating a good biological activity of the ribozyme. Moreover, this reduction in VEGF secretion had the important functional consequence of drastically diminishing the formation of tube-like structures of human umbilical vascular endothelial cells in a Matrigel in vitro angiogenesis assay. In conclusion, our VAI-embedded anti-VEGF ribozyme is a good inhibitor of angiogenesis in vitro, in a glioblastoma cell context. Thus, it may represent a useful tool for future applications in vivo, for antiangiogenic gene therapy of glioblastoma and of highly vascularized tumors. Copyright 2004 S. Karger AG, Basel

Growth phase-dependent control of R27 conjugation is mediated by the interplay between the plasmid-encoded regulatory circuit TrhR/TrhY-HtdA and the cAMP regulon.

PubMed

Gibert, Marta; Paytubi, Sonia; Beltrán, Sergi; Juárez, Antonio; Balsalobre, Carlos; Madrid, Cristina

2016-12-01

Plasmids of the incompatibility group HI1 (IncHI1) have been isolated from several Gram-negative pathogens and are associated with the spread of multidrug resistance. Their conjugation is tightly regulated and it is inhibited at temperatures higher than 30°C, indicating that conjugation occurs outside warm-blooded hosts. Using R27, the prototype of IncHI1 plasmids, we report that plasmid transfer efficiency in E. coli strongly depends on the physiological state of the donor cells. Conjugation frequency is high when cells are actively growing, dropping sharply when cells enter the stationary phase of growth. Accordingly, our transcriptomic assays show significant downregulation of numerous R27 genes during the stationary phase, including several tra (transfer) genes. Growth phase-dependent regulation of tra genes transcription is independent of H-NS, a silencer of horizontal gene transfer, and ppGpp and RpoS, regulators of the stationary phase, but highly dependent on the plasmid-encoded regulatory circuit TrhR/TrhY-HtdA. The metabolic sensor cAMP, whose synthesis is chromosomally encoded, is also involved in the growth phase regulation of R27 conjugation by modulating htdA expression. Our data suggest that the involvement of regulators encoded by both chromosome and plasmid are required for efficient physiological control of IncHI1 plasmid conjugation. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

TonB-Dependent Transporters Expressed by Neisseria gonorrhoeae

PubMed Central

Cornelissen, Cynthia Nau; Hollander, Aimee

2011-01-01

Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention. PMID:21747812

Biological properties of human skeletal myoblasts genetically modified to simultaneously overexpress the pro-angiogenic factors vascular endothelial growth factor-A and fibroblast growth factor-4.

PubMed

Zimna, A; Janeczek, A; Rozwadowska, N; Fraczek, M; Kucharzewska, P; Rucinski, M; Mietkiewski, T; Kurpisz, M

2014-04-01

Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities. We used a bicistronic plasmid vector encoding two factors introduced via an efficient myoblast electroporation method. The levels of overexpressed proteins were assessed, and their functionality at capillary formation was evaluated. This combined approach led to a high level of non-viral transient overexpression of both pro-angiogenic proteins, which proved to be potent regulators of blood vessel development assayed in capillary formation tests. We demonstrated in in vitro conditions that the transfection of human skeletal myoblasts with both FGF-4 and VEGF did not affect their basic biological properties such as the cell cycle, proliferation or expression of myogenic lineage-specific genes, and the modified cells adapted to oxidative stress conditions. Overall, the results obtained suggest that the applied combined approach with the use of two pro-angiogenic genes overexpressed in skeletal muscle stem cells may be an interesting alternative for the effective therapy of myocardial infarction in animal models and/or prospective clinical trials.

Large-scale network integration in the human brain tracks temporal fluctuations in memory encoding performance.

PubMed

Keerativittayayut, Ruedeerat; Aoki, Ryuta; Sarabi, Mitra Taghizadeh; Jimura, Koji; Nakahara, Kiyoshi

2018-06-18

Although activation/deactivation of specific brain regions have been shown to be predictive of successful memory encoding, the relationship between time-varying large-scale brain networks and fluctuations of memory encoding performance remains unclear. Here we investigated time-varying functional connectivity patterns across the human brain in periods of 30-40 s, which have recently been implicated in various cognitive functions. During functional magnetic resonance imaging, participants performed a memory encoding task, and their performance was assessed with a subsequent surprise memory test. A graph analysis of functional connectivity patterns revealed that increased integration of the subcortical, default-mode, salience, and visual subnetworks with other subnetworks is a hallmark of successful memory encoding. Moreover, multivariate analysis using the graph metrics of integration reliably classified the brain network states into the period of high (vs. low) memory encoding performance. Our findings suggest that a diverse set of brain systems dynamically interact to support successful memory encoding. © 2018, Keerativittayayut et al.

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection

PubMed Central

De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain

2016-01-01

ABSTRACT Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae. Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum. IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete’s foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae. Comparing gene expression during infection on guinea pigs with keratin degradation in vitro, which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo, encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates. PMID:27822542

RNA Sequencing-Based Genome Reannotation of the Dermatophyte Arthroderma benhamiae and Characterization of Its Secretome and Whole Gene Expression Profile during Infection.

PubMed

Tran, Van Du T; De Coi, Niccolò; Feuermann, Marc; Schmid-Siegert, Emanuel; Băguţ, Elena-Tatiana; Mignon, Bernard; Waridel, Patrice; Peter, Corinne; Pradervand, Sylvain; Pagni, Marco; Monod, Michel

2016-01-01

Dermatophytes are the most common agents of superficial mycoses in humans and animals. The aim of the present investigation was to systematically identify the extracellular, possibly secreted, proteins that are putative virulence factors and antigenic molecules of dermatophytes. A complete gene expression profile of Arthroderma benhamiae was obtained during infection of its natural host (guinea pig) using RNA sequencing (RNA-seq) technology. This profile was completed with those of the fungus cultivated in vitro in two media containing either keratin or soy meal protein as the sole source of nitrogen and in Sabouraud medium. More than 60% of transcripts deduced from RNA-seq data differ from those previously deposited for A. benhamiae . Using these RNA-seq data along with an automatic gene annotation procedure, followed by manual curation, we produced a new annotation of the A. benhamiae genome. This annotation comprised 7,405 coding sequences (CDSs), among which only 2,662 were identical to the currently available annotation, 383 were newly identified, and 15 secreted proteins were manually corrected. The expression profile of genes encoding proteins with a signal peptide in infected guinea pigs was found to be very different from that during in vitro growth when using keratin as the substrate. Especially, the sets of the 12 most highly expressed genes encoding proteases with a signal sequence had only the putative vacuolar aspartic protease gene PEP2 in common, during infection and in keratin medium. The most upregulated gene encoding a secreted protease during infection was that encoding subtilisin SUB6, which is a known major allergen in the related dermatophyte Trichophyton rubrum . IMPORTANCE Dermatophytoses (ringworm, jock itch, athlete's foot, and nail infections) are the most common fungal infections, but their virulence mechanisms are poorly understood. Combining transcriptomic data obtained from growth under various culture conditions with data obtained during infection led to a significantly improved genome annotation. About 65% of the protein-encoding genes predicted with our protocol did not match the existing annotation for A. benhamiae . Comparing gene expression during infection on guinea pigs with keratin degradation in vitro , which is supposed to mimic the host environment, revealed the critical importance of using real in vivo conditions for investigating virulence mechanisms. The analysis of genes expressed in vivo , encoding cell surface and secreted proteins, particularly proteases, led to the identification of new allergen and virulence factor candidates.

Two Closely Related Genes of Arabidopsis Encode Plastidial Cytidinediphosphate Diacylglycerol Synthases Essential for Photoautotrophic Growth1[C

PubMed Central

Haselier, André; Akbari, Hana; Weth, Agnes; Baumgartner, Werner; Frentzen, Margrit

2010-01-01

Cytidinediphosphate diacylglycerol synthase (CDS) catalyzes the formation of cytidinediphosphate diacylglycerol, an essential precursor of anionic phosphoglycerolipids like phosphatidylglycerol or -inositol. In plant cells, CDS isozymes are located in plastids, mitochondria, and microsomes. Here, we show that these isozymes are encoded by five genes in Arabidopsis (Arabidopsis thaliana). Alternative translation initiation or alternative splicing of CDS2 and CDS4 transcripts can result in up to 10 isoforms. Most of the cDNAs encoding the various plant isoforms were functionally expressed in yeast and rescued the nonviable phenotype of the mutant strain lacking CDS activity. The closely related genes CDS4 and CDS5 were found to encode plastidial isozymes with similar catalytic properties. Inactivation of both genes was required to obtain Arabidopsis mutant lines with a visible phenotype, suggesting that the genes have redundant functions. Analysis of these Arabidopsis mutants provided further independent evidence for the importance of plastidial phosphatidylglycerol for structure and function of thylakoid membranes and, hence, for photoautotrophic growth. PMID:20442275

More Genetic Engineering With Cloned Hemoglobin Genes

NASA Technical Reports Server (NTRS)

Bailey, James E.

1992-01-01

Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

PubMed Central

Dialynas, D P; Murre, C; Quertermous, T; Boss, J M; Leiden, J M; Seidman, J G; Strominger, J L

1986-01-01

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation. Images PMID:3458221

Spiking, Bursting, and Population Dynamics in a Network of Growth Transform Neurons.

PubMed

Gangopadhyay, Ahana; Chakrabartty, Shantanu

2018-06-01

This paper investigates the dynamical properties of a network of neurons, each of which implements an asynchronous mapping based on polynomial growth transforms. In the first part of this paper, we present a geometric approach for visualizing the dynamics of the network where each of the neurons traverses a trajectory in a dual optimization space, whereas the network itself traverses a trajectory in an equivalent primal optimization space. We show that as the network learns to solve basic classification tasks, different choices of primal-dual mapping produce unique but interpretable neural dynamics like noise shaping, spiking, and bursting. While the proposed framework is general enough, in this paper, we demonstrate its use for designing support vector machines (SVMs) that exhibit noise-shaping properties similar to those of modulators, and for designing SVMs that learn to encode information using spikes and bursts. It is demonstrated that the emergent switching, spiking, and burst dynamics produced by each neuron encodes its respective margin of separation from a classification hyperplane whose parameters are encoded by the network population dynamics. We believe that the proposed growth transform neuron model and the underlying geometric framework could serve as an important tool to connect well-established machine learning algorithms like SVMs to neuromorphic principles like spiking, bursting, population encoding, and noise shaping.

Lessons on the pathogenesis of aneurysm from heritable conditions

PubMed Central

Lindsay, Mark E.; Dietz, Harry C.

2013-01-01

Aortic aneurysm is common, accounting for 1–2% of all deaths in industrialized countries. Early theories of the causes of human aneurysm mostly focused on inherited or acquired defects in components of the extracellular matrix in the aorta. Although several mutations in the genes encoding extracellular matrix proteins have been recognized, more recent discoveries have shown important perturbations in cytokine signalling cascades and intracellular components of the smooth muscle contractile apparatus. The modelling of single-gene heritable aneurysm disorders in mice has shown unexpected involvement of the transforming growth factor-β cytokine pathway in aortic aneurysm, highlighting the potential for new therapeutic strategies. PMID:21593863

The ENCODE project: implications for psychiatric genetics.

PubMed

Kavanagh, D H; Dwyer, S; O'Donovan, M C; Owen, M J

2013-05-01

The ENCyclopedia Of DNA Elements (ENCODE) project is a public research consortium that aims to identify all functional elements of the human genome sequence. The project comprised 1640 data sets, from 147 different cell type and the findings were released in a coordinated set of 34 publications across several journals. The ENCODE publications report that 80.4% of the human genome displays some functionality. These data have important implications for interpreting results from large-scale genetics studies. We reviewed some of the key findings from the ENCODE publications and discuss how they can influence or inform further investigations into the genetic factors contributing to neuropsychiatric disorders.

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

PubMed Central

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

1993-01-01

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

PDGF-B Gene Therapy Accelerates Bone Engineering and Oral Implant Osseointegration

PubMed Central

Chang, Po-Chun; Seol, Yang-Jo; Cirelli, Joni A; Pellegrini, Gaia R.; Jin, Qiming; Franco, Lea M.; Goldstein, Steven A.; Chandler, Lois A.; Sosnowski, Barbara; Giannobile, William V.

2009-01-01

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due in part to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5×108 or 5.5×109 pfu/ml), Ad encoding luciferase (Ad-Luc; 5.5×109 pfu/ml; control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg/ml). Bone repair and osseointegration were measured via backscattered SEM, histomorphometry, microcomputed tomography, and biomechanical assessments. Further, a panel of local and systemic safety assessments was performed. Results demonstrated bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared to Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B demonstrates regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable to rhPDGF-BB protein delivery in vivo. PMID:19741730

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila.

PubMed

Baril, Caroline; Gavory, Gwenaëlle; Bidla, Gawa; Knævelsrud, Helene; Sauvageau, Guy; Therrien, Marc

2017-01-01

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings. Copyright © 2016 Elsevier Inc. All rights reserved.

Cellodextrin utilization by bifidobacterium breve UCC2003.

PubMed

Pokusaeva, Karina; O'Connell-Motherway, Mary; Zomer, Aldert; Macsharry, John; Fitzgerald, Gerald F; van Sinderen, Douwe

2011-03-01

Cellodextrins, the incomplete hydrolysis products from insoluble cellulose, are accessible as a carbon source to certain members of the human gut microbiota, such as Bifidobacterium breve UCC2003. Transcription of the cldEFGC gene cluster of B. breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating this cluster in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this commensal. Moreover, our results suggest that transcription of the cld cluster is controlled by a LacI-type regulator encoded by cldR, located immediately upstream of cldE. Gel mobility shift assays using purified CldR(His) (produced by the incorporation of a His(12)-encoding sequence into the 3' end of the cldC gene) indicate that the cldEFGC promoter is subject to negative control by CldR(His), which binds to two inverted repeats. Analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose, and cellopentaose, with cellotriose apparently representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 is, to the best of our knowledge, the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity toward various cellodextrins.

Cancer Risk and Eicosanoid Production: Interaction between the Protective Effect of Long Chain Omega-3 Polyunsaturated Fatty Acid Intake and Genotype

PubMed Central

Lenihan-Geels, Georgia; Bishop, Karen S.; Ferguson, Lynnette R.

2016-01-01

Dietary inclusion of fish and fish supplements as a means to improve cancer prognosis and prevent tumour growth is largely controversial. Long chain omega-3 polyunsaturated fatty acids (LCn-3 PUFA), eicosapentaenoic acid and docosahexaenoic acid, may modulate the production of inflammatory eicosanoids, thereby influencing local inflammatory status, which is important in cancer development. Although in vitro studies have demonstrated inhibition of tumour cell growth and proliferation by LCn-3 PUFA, results from human studies have been mainly inconsistent. Genes involved in the desaturation of fatty acids, as well as the genes encoding enzymes responsible for eicosanoid production, are known to be implicated in tumour development. This review discusses the current evidence for an interaction between genetic polymorphisms and dietary LCn-3 PUFA in the risk for breast, prostate and colorectal cancers, in regards to inflammation and eicosanoid synthesis. PMID:26891335

Battle against cancer: an everlasting saga of p53.

PubMed

Hao, Qian; Cho, William C

2014-12-01

Cancer is one of the most life-threatening diseases characterized by uncontrolled growth and spread of malignant cells. The tumor suppressor p53 is the master regulator of tumor cell growth and proliferation. In response to various stress signals, p53 can be activated and transcriptionally induces a myriad of target genes, including both protein-encoding and non-coding genes, controlling cell cycle progression, DNA repair, senescence, apoptosis, autophagy and metabolism of tumor cells. However, around 50% of human cancers harbor mutant p53 and, in the majority of the remaining cancers, p53 is inactivated through multiple mechanisms. Herein, we review the recent progress in understanding the molecular basis of p53 signaling, particularly the newly identified ribosomal stress-p53 pathway, and the development of chemotherapeutics via activating wild-type p53 or restoring mutant p53 functions in cancer. A full understanding of p53 regulation will aid the development of effective cancer treatments.

Sensory deprivation in Staphylococcus aureus.

PubMed

Villanueva, Maite; García, Begoña; Valle, Jaione; Rapún, Beatriz; Ruiz de Los Mozos, Igor; Solano, Cristina; Martí, Miguel; Penadés, José R; Toledo-Arana, Alejandro; Lasa, Iñigo

2018-02-06

Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches.

The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon▿

PubMed Central

Graef, Katy M.; Vreede, Frank T.; Lau, Yuk-Fai; McCall, Amber W.; Carr, Simon M.; Subbarao, Kanta; Fodor, Ervin

2010-01-01

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence. PMID:20538852

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

PubMed

Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Stress as a mnemonic filter: Interactions between medial temporal lobe encoding processes and post-encoding stress

PubMed Central

Ritchey, Maureen; McCullough, Andrew M.; Ranganath, Charan; Yonelinas, Andrew P.

2016-01-01

Acute stress has been shown to modulate memory for recently learned information, an effect attributed to the influence of stress hormones on medial temporal lobe (MTL) consolidation processes. However, little is known about which memories will be affected when stress follows encoding. One possibility is that stress interacts with encoding processes to selectively protect memories that had elicited responses in the hippocampus and amygdala, two MTL structures important for memory formation. There is limited evidence for interactions between encoding processes and consolidation effects in humans, but recent studies of consolidation in rodents have emphasized the importance of encoding “tags” for determining the impact of consolidation manipulations on memory. Here, we used fMRI in humans to test the hypothesis that the effects of post-encoding stress depend on MTL processes observed during encoding. We found that changes in stress hormone levels were associated with an increase in the contingency of memory outcomes on hippocampal and amygdala encoding responses. That is, for participants showing high cortisol reactivity, memories became more dependent on MTL activity observed during encoding, thereby shifting the distribution of recollected events toward those that had elicited relatively high activation. Surprisingly, this effect was generally larger for neutral, compared to emotionally negative, memories. The results suggest that stress does not uniformly enhance memory, but instead selectively preserves memories tagged during encoding, effectively acting as mnemonic filter. PMID:27774683

Nucleic acids encoding mosaic clade M human immunodeficiency virus type 1 (HIV-1) envelope immunogens

DOEpatents

Korber, Bette T; Fischer, William; Liao, Hua-Xin; Haynes, Barton F; Letvin, Norman; Hahn, Beatrice H

2015-04-21

The present invention relates to nucleic acids encoding mosaic clade M HIV-1 Env polypeptides and to compositions and vectors comprising same. The nucleic acids of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

Comparison of H.265/HEVC encoders

NASA Astrophysics Data System (ADS)

Trochimiuk, Maciej

2016-09-01

The H.265/HEVC is the state-of-the-art video compression standard, which allows the bitrate reduction up to 50% compared with its predecessor, H.264/AVC, maintaining equal perceptual video quality. The growth in coding efficiency was achieved by increasing the number of available intra- and inter-frame prediction features and improvements in existing ones, such as entropy encoding and filtering. Nevertheless, to achieve real-time performance of the encoder, simplifications in algorithm are inevitable. Some features and coding modes shall be skipped, to reduce time needed to evaluate modes forwarded to rate-distortion optimisation. Thus, the potential acceleration of the encoding process comes at the expense of coding efficiency. In this paper, a trade-off between video quality and encoding speed of various H.265/HEVC encoders is discussed.

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

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

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply

DTIC Science & Technology

2006-05-01

W81XWH-04-1-0489 TITLE: An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply PRINCIPAL...Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply 5b. GRANT NUMBER W81XWH-04-1-0489 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...blocking renewal of blood vessel growth in the tumor bed, have been proposed as suitable antitumor strategies. Endoglin (CD105) is a suitable

Networks for image acquisition, processing and display

NASA Technical Reports Server (NTRS)

Ahumada, Albert J., Jr.

1990-01-01

The human visual system comprises layers of networks which sample, process, and code images. Understanding these networks is a valuable means of understanding human vision and of designing autonomous vision systems based on network processing. Ames Research Center has an ongoing program to develop computational models of such networks. The models predict human performance in detection of targets and in discrimination of displayed information. In addition, the models are artificial vision systems sharing properties with biological vision that has been tuned by evolution for high performance. Properties include variable density sampling, noise immunity, multi-resolution coding, and fault-tolerance. The research stresses analysis of noise in visual networks, including sampling, photon, and processing unit noises. Specific accomplishments include: models of sampling array growth with variable density and irregularity comparable to that of the retinal cone mosaic; noise models of networks with signal-dependent and independent noise; models of network connection development for preserving spatial registration and interpolation; multi-resolution encoding models based on hexagonal arrays (HOP transform); and mathematical procedures for simplifying analysis of large networks.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

PubMed

Gui, Tao; Zhang, Meiling; Chen, Jianwen; Zhang, Yuanliang; Zhou, Naru; Zhang, Yu; Tao, Jia; Sui, Liucai; Li, Yunsheng; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

2012-08-01

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Trastuzumab-binding peptide display by Tobacco mosaic virus

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

Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.

2010-11-10

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturabilitymore » of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.« less

Characterization of the canine mda-7 gene, transcripts and expression patterns

PubMed Central

Sandey, Maninder; Bird, R. Curtis; Das, Swadesh K.; Sarkar, Devanand; Curiel, David T.; Fisher, Paul B.; Smith, Bruce F.

2014-01-01

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5′-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog. PMID:24865935

The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model.

PubMed

Mirzaei, Hamed; Salehi, Hossein; Oskuee, Reza Kazemi; Mohammadpour, Ali; Mirzaei, Hamid Reza; Sharifi, Mohammad Reza; Salarinia, Reza; Darani, Hossein Yousofi; Mokhtari, Mojgan; Masoudifar, Aria; Sahebkar, Amirhossein; Salehi, Rasoul; Jaafari, Mahmoud Reza

2018-04-10

Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC ex vivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to in vivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences.

PubMed

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-06-09

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

Inhibition of herpes simplex virus 1 gene expression and replication by RNase P-associated external guide sequences

PubMed Central

Liu, Jin; Shao, Luyao; Trang, Phong; Yang, Zhu; Reeves, Michael; Sun, Xu; Vu, Gia-Phong; Wang, Yu; Li, Hongjian; Zheng, Congyi; Lu, Sangwei; Liu, Fenyong

2016-01-01

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics. PMID:27279482

Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures.

PubMed

Fox, Emily P; Cowley, Elise S; Nobile, Clarissa J; Hartooni, Nairi; Newman, Dianne K; Johnson, Alexander D

2014-10-20

The human microbiome contains diverse microorganisms, which share and compete for the same environmental niches. A major microbial growth form in the human body is the biofilm state, where tightly packed bacterial, archaeal, and fungal cells must cooperate and/or compete for resources in order to survive. We examined mixed biofilms composed of the major fungal species of the gut microbiome, Candida albicans, and each of five prevalent bacterial gastrointestinal inhabitants: Bacteroides fragilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. We observed that biofilms formed by C. albicans provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found that coculture with bacteria in biofilms induces massive gene expression changes in C. albicans, including upregulation of WOR1, which encodes a transcription regulator that controls a phenotypic switch in C. albicans, from the "white" cell type to the "opaque" cell type. Finally, we observed that in suspension cultures, C. perfringens induces aggregation of C. albicans into "mini-biofilms," which allow C. perfringens cells to survive in a normally toxic environment. This work indicates that bacteria and C. albicans interactions modulate the local chemistry of their environment in multiple ways to create niches favorable to their growth and survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

Parametric fMRI analysis of visual encoding in the human medial temporal lobe.

PubMed

Rombouts, S A; Scheltens, P; Machielson, W C; Barkhof, F; Hoogenraad, F G; Veltman, D J; Valk, J; Witter, M P

1999-01-01

A number of functional brain imaging studies indicate that the medial temporal lobe system is crucially involved in encoding new information into memory. However, most studies were based on differences in brain activity between encoding of familiar vs. novel stimuli. To further study the underlying cognitive processes, we applied a parametric design of encoding. Seven healthy subjects were instructed to encode complex color pictures into memory. Stimuli were presented in a parametric fashion at different rates, thus representing different loads of encoding. Functional magnetic resonance imaging (fMRI) was used to assess changes in brain activation. To determine the number of pictures successfully stored into memory, recognition scores were determined afterwards. During encoding, brain activation occurred in the medial temporal lobe, comparable to the results obtained by others. Increasing the encoding load resulted in an increase in the number of successfully stored items. This was reflected in a significant increase in brain activation in the left lingual gyrus, in the left and right parahippocampal gyrus, and in the right inferior frontal gyrus. This study shows that fMRI can detect changes in brain activation during variation of one aspect of higher cognitive tasks. Further, it strongly supports the notion that the human medial temporal lobe is involved in encoding novel visual information into memory.

The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope

PubMed Central

Shikhagaie, Medya; Mercé-Maldonado, Eva; Isern, Elena; Muntasell, Aura; Albà, M. Mar; López-Botet, Miguel; Hengel, Hartmut

2012-01-01

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism. PMID:22345456

Encoding and Decoding Models in Cognitive Electrophysiology

PubMed Central

Holdgraf, Christopher R.; Rieger, Jochem W.; Micheli, Cristiano; Martin, Stephanie; Knight, Robert T.; Theunissen, Frederic E.

2017-01-01

Cognitive neuroscience has seen rapid growth in the size and complexity of data recorded from the human brain as well as in the computational tools available to analyze this data. This data explosion has resulted in an increased use of multivariate, model-based methods for asking neuroscience questions, allowing scientists to investigate multiple hypotheses with a single dataset, to use complex, time-varying stimuli, and to study the human brain under more naturalistic conditions. These tools come in the form of “Encoding” models, in which stimulus features are used to model brain activity, and “Decoding” models, in which neural features are used to generated a stimulus output. Here we review the current state of encoding and decoding models in cognitive electrophysiology and provide a practical guide toward conducting experiments and analyses in this emerging field. Our examples focus on using linear models in the study of human language and audition. We show how to calculate auditory receptive fields from natural sounds as well as how to decode neural recordings to predict speech. The paper aims to be a useful tutorial to these approaches, and a practical introduction to using machine learning and applied statistics to build models of neural activity. The data analytic approaches we discuss may also be applied to other sensory modalities, motor systems, and cognitive systems, and we cover some examples in these areas. In addition, a collection of Jupyter notebooks is publicly available as a complement to the material covered in this paper, providing code examples and tutorials for predictive modeling in python. The aim is to provide a practical understanding of predictive modeling of human brain data and to propose best-practices in conducting these analyses. PMID:29018336

A decade of human genome project conclusion: Scientific diffusion about our genome knowledge.

PubMed

Moraes, Fernanda; Góes, Andréa

2016-05-06

The Human Genome Project (HGP) was initiated in 1990 and completed in 2003. It aimed to sequence the whole human genome. Although it represented an advance in understanding the human genome and its complexity, many questions remained unanswered. Other projects were launched in order to unravel the mysteries of our genome, including the ENCyclopedia of DNA Elements (ENCODE). This review aims to analyze the evolution of scientific knowledge related to both the HGP and ENCODE projects. Data were retrieved from scientific articles published in 1990-2014, a period comprising the development and the 10 years following the HGP completion. The fact that only 20,000 genes are protein and RNA-coding is one of the most striking HGP results. A new concept about the organization of genome arose. The ENCODE project was initiated in 2003 and targeted to map the functional elements of the human genome. This project revealed that the human genome is pervasively transcribed. Therefore, it was determined that a large part of the non-protein coding regions are functional. Finally, a more sophisticated view of chromatin structure emerged. The mechanistic functioning of the genome has been redrafted, revealing a much more complex picture. Besides, a gene-centric conception of the organism has to be reviewed. A number of criticisms have emerged against the ENCODE project approaches, raising the question of whether non-conserved but biochemically active regions are truly functional. Thus, HGP and ENCODE projects accomplished a great map of the human genome, but the data generated still requires further in depth analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:215-223, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

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

Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activatedmore » GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.« less

Word Order and Voice Influence the Timing of Verb Planning in German Sentence Production.

PubMed

Sauppe, Sebastian

2017-01-01

Theories of incremental sentence production make different assumptions about when speakers encode information about described events and when verbs are selected, accordingly. An eye tracking experiment on German testing the predictions from linear and hierarchical incrementality about the timing of event encoding and verb planning is reported. In the experiment, participants described depictions of two-participant events with sentences that differed in voice and word order. Verb-medial active sentences and actives and passives with sentence-final verbs were compared. Linear incrementality predicts that sentences with verbs placed early differ from verb-final sentences because verbs are assumed to only be planned shortly before they are articulated. By contrast, hierarchical incrementality assumes that speakers start planning with relational encoding of the event. A weak version of hierarchical incrementality assumes that only the action is encoded at the outset of formulation and selection of lexical verbs only occurs shortly before they are articulated, leading to the prediction of different fixation patterns for verb-medial and verb-final sentences. A strong version of hierarchical incrementality predicts no differences between verb-medial and verb-final sentences because it assumes that verbs are always lexically selected early in the formulation process. Based on growth curve analyses of fixations to agent and patient characters in the described pictures, and the influence of character humanness and the lack of an influence of the visual salience of characters on speakers' choice of active or passive voice, the current results suggest that while verb planning does not necessarily occur early during formulation, speakers of German always create an event representation early.

Anti-Epidermal Growth Factor Receptor Gene Therapy for Glioblastoma

PubMed Central

Hicks, Martin J.; Chiuchiolo, Maria J.; Ballon, Douglas; Dyke, Jonathan P.; Aronowitz, Eric; Funato, Kosuke; Tabar, Viviane; Havlicek, David; Fan, Fan; Sondhi, Dolan; Kaminsky, Stephen M.; Crystal, Ronald G.

2016-01-01

Glioblastoma multiforme (GBM) is the most common and aggressive primary intracranial brain tumor in adults with a mean survival of 14 to 15 months. Aberrant activation of the epidermal growth factor receptor (EGFR) plays a significant role in GBM progression, with amplification or overexpression of EGFR in 60% of GBM tumors. To target EGFR expressed by GBM, we have developed a strategy to deliver the coding sequence for cetuximab, an anti-EGFR antibody, directly to the CNS using an adeno-associated virus serotype rh.10 gene transfer vector. The data demonstrates that single, local delivery of an anti-EGFR antibody by an AAVrh.10 vector coding for cetuximab (AAVrh.10Cetmab) reduces GBM tumor growth and increases survival in xenograft mouse models of a human GBM EGFR-expressing cell line and patient-derived GBM. AAVrh10.CetMab-treated mice displayed a reduction in cachexia, a significant decrease in tumor volume and a prolonged survival following therapy. Adeno-associated-directed delivery of a gene encoding a therapeutic anti-EGFR monoclonal antibody may be an effective strategy to treat GBM. PMID:27711187

Protein Hydrolysates from Non-bovine and Plant Sources Replaces Tryptone in Microbiological Media

NASA Astrophysics Data System (ADS)

Ranganathan, Yamini; Patel, Shifa; Pasupuleti, Vijai K.; Meganathan, R.

Tryptone (pancreatic digest of casein) is a common ingredient in laboratory and fermentation media for growing wild-type and genetically modified microorganisms. Many of the commercially manufactured products such as human growth hormone, antibiotics, insulin, etc. are produced by recombinant strains grown on materials derived from bovine sources. With the emergence of Bovine Spongiform Encephalopathy (BSE) and the consequent increase in Food and Drug Administration (FDA) regulations, elimination of materials of bovine origin from fermentation media is of paramount importance. To achieve this objective, a number of protein hydrolysates derived from non-bovine animal and plant sources were evaluated. Tryptone in Luria-Bertani (LB) broth was replaced with an equal quantity of alternate protein hydrolysates. Four of the six hydrolysates (one animal and three from plants) were found to efficiently replace the tryptone present in LB-medium as measured by growth rate and growth yield of a recombinant Escherichia coli strain. In addition, we have determined plasmid stability, inducibility and activity of the plasmid encoded β-galactosidase in the recombinant strain grown in the presence of various protein hydrolysates.

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride

PubMed Central

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E.; Staege, Martin S.; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS. PMID:29515560

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

PubMed

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E; Staege, Martin S; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

Four regulatory elements in the human c-fos promoter mediate transactivation by HTLV-1 Tax protein.

PubMed

Alexandre, C; Verrier, B

1991-04-01

Expression of the human c-fos proto-oncogene is activated in trans by the Tax protein encoded by human T-cell leukemia virus type-1 (HTLV-1). Indeed, we show here that a HeLa clone stably transfected by Tax expresses Fos at a high level. We also show that multiple elements of the human c-fos promoter, i.e. the v-sis conditioned medium inducible element (SIE), the dyad symmetry element (DSE) necessary for growth factor induction, the octanucleotide direct repeat element (DR), and the cyclic AMP response element (CRE) centred at -60, can all mediate Tax transactivation. In the DSE, the 10bp central core that binds the serum response factor (SRF) is, by itself, sufficient to mediate Tax transactivation. Moreover, a CRE-binding protein is involved in Tax activation through the CRE-60 element. Since Fos is a transregulator of cellular genes, our results suggest that the oncoprotein plays a crucial role in T-cell transformation by HTLV-1 in conjunction with other Tax-inducible genes.

Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation

PubMed Central

Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P.; Eaves, Connie J.

2015-01-01

Summary Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44+NGFR+ cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

Pathobiologic Roles of Epstein–Barr Virus-Encoded MicroRNAs in Human Lymphomas

PubMed Central

Navari, Mohsen; Etebari, Maryam; Ibrahimi, Mostafa; Leoncini, Lorenzo

2018-01-01

Epstein–Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs—small RNAs with a posttranscriptional regulating role—are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas. PMID:29649101

[Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells].

PubMed

Shang, Shu-huan; Zhang, Yu-feng; Shi, Bin; Cheng, Xiang-rong

2008-10-01

To construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC). The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01. Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

PubMed

Fairley, D J; Wang, G; Rensing, C; Pepper, I L; Larkin, M J

2006-12-01

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

PubMed Central

Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

2005-01-01

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection. PMID:16163387

The pbrB gene encodes a laccase required for DHN-melanin synthesis in conidia of Talaromyces (Penicillium) marneffei.

PubMed

Sapmak, Ariya; Boyce, Kylie J; Andrianopoulos, Alex; Vanittanakom, Nongnuch

2015-01-01

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.

The pbrB Gene Encodes a Laccase Required for DHN-Melanin Synthesis in Conidia of Talaromyces (Penicillium) marneffei

PubMed Central

Sapmak, Ariya; Boyce, Kylie J.; Andrianopoulos, Alex; Vanittanakom, Nongnuch

2015-01-01

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type. PMID:25866870

Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques

PubMed Central

Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

2014-01-01

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environments. V. parahaemolyticus is the leading causal agent of human acute gastroenteritis following the consumption of raw, undercooked, or mishandled marine products. In rare cases, V. parahaemolyticus causes wound infection, ear infection or septicaemia in individuals with pre-existing medical conditions. V. parahaemolyticus has two hemolysins virulence factors that are thermostable direct hemolysin (tdh)-a pore-forming protein that contributes to the invasiveness of the bacterium in humans, and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. In addition, the bacterium is also encodes for adhesions and type III secretion systems (T3SS1 and T3SS2) to ensure its survival in the environment. This review aims at discussing the V. parahaemolyticus growth and characteristics, pathogenesis, prevalence and advances in molecular identification techniques. PMID:25566219

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

PubMed

Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

1990-01-01

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Neisseria gonorrhoeae Modulates Iron-Limiting Innate Immune Defenses in Macrophages

PubMed Central

Zughaier, Susu M.; Kandler, Justin L.; Shafer, William M.

2014-01-01

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. The gonococcus can survive extracellularly and intracellularly, but in both environments the bacteria must acquire iron from host proteins for survival. However, upon infection the host uses a defensive response by limiting the bioavailability of iron by a number of mechanisms including the enhanced expression of hepcidin, the master iron-regulating hormone, which reduces iron uptake from the gut and retains iron in macrophages. The host also secretes the antibacterial protein NGAL, which sequesters bacterial siderophores and therefore inhibits bacterial growth. To learn whether intracellular gonococci can subvert this defensive response, we examined expression of host genes that encode proteins involved in modulating levels of intracellular iron. We found that N. gonorrhoeae can survive in association (tightly adherent and intracellular) with monocytes and macrophages and upregulates a panel of its iron-responsive genes in this environment. We also found that gonococcal infection of human monocytes or murine macrophages resulted in the upregulation of hepcidin, NGAL, and NRAMP1 as well as downregulation of the expression of the gene encoding the short chain 3-hydroxybutyrate dehydrogenase (BDH2); BDH2 catalyzes the production of the mammalian siderophore 2,5-DHBA involved in chelating and detoxifying iron. Based on these findings, we propose that N. gonorrhoeae can subvert the iron-limiting innate immune defenses to facilitate iron acquisition and intracellular survival. PMID:24489950

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

PubMed

Gaderer, Romana; Seidl-Seiboth, Verena; de Vries, Ronald P; Seiboth, Bernhard; Kappel, Lisa

2017-10-01

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretin receptor superfamily with an unusual extracellular domain

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

Hamann, J.; Hamann, D.; Lier, R.A.W.

1995-08-15

CD97 is a monomeric glycoprotein of 75 to 85 kDa that is induced rapidly on the surface of most leukocytes upon activation. We herein report the isolation of a cDNA encoding human CD97 by expression cloning in COS cells. The 3-kb cDNA clone encodes a mature polypeptide chain of 722 amino acids with a predicted molecular mass of 79 kDa. Within the C-terminal part of the protein, a region with seven hydrophobic segments was identified, suggesting that CD97 is a seven-span transmembrane molecule. Sequence comparison indicates that CD97 is the first leukocyte Ag in a recently described superfamily that includesmore » the receptors for secretin, calcitonin, and other mammalian and insect peptide hormones. Different from these receptors, CD97 has an extended extracellular region of 433 amino acids that possesses three N-terminal epidermal growth factor-like domains, two of them with a calcium-binding site, and single Arg-Gly-Asp (RGD) motif. The existence of structural elements characteristic for extracellular matrix proteins in a seven-span transmembrane molecule makes CD97 a receptor potentially involved in both adhesion and signaling processes early after leukocyte activation. The gene encoding CD97 is localized on chromosome 19 (19p13.12-13.2).« less

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

A direct link between carbohydrate utilization and virulence in the major human pathogen group A Streptococcus.

PubMed

Shelburne, Samuel A; Keith, David; Horstmann, Nicola; Sumby, Paul; Davenport, Michael T; Graviss, Edward A; Brennan, Richard G; Musser, James M

2008-02-05

Although central to pathogenesis, the molecular mechanisms used by microbes to regulate virulence factor production in specific environments during host-pathogen interaction are poorly defined. Several recent ex vivo and in vivo studies have found that the level of group A Streptococcus (GAS) virulence factor gene transcripts is temporally related to altered expression of genes encoding carbohydrate utilization proteins. These findings stimulated us to analyze the role in pathogenesis of catabolite control protein A (CcpA), a GAS ortholog of a key global regulator of carbohydrate metabolism in Bacillus subtilis. Inasmuch as the genomewide effects of CcpA in a human pathogen are unknown, we analyzed the transcriptome of a DeltaccpA isogenic mutant strain grown in nutrient-rich medium. CcpA influences the transcript levels of many carbohydrate utilization genes and several well characterized GAS virulence factors, including the potent cytolysin streptolysin S. Compared with the wild-type parental strain, the DeltaccpA isogenic mutant strain was significantly less virulent in a mouse model of invasive infection. Moreover, the isogenic mutant strain was significantly impaired in ability to colonize the mouse oropharynx. When grown in human saliva, a nutrient-limited environment, CcpA influenced production of several key virulence factors not influenced during growth in nutrient-rich medium. Purified recombinant CcpA bound to the promoter region of the gene encoding streptolysin S. Our discovery that GAS virulence and complex carbohydrate utilization are directly linked through CcpA provides enhanced understanding of a mechanism used by a Gram-positive pathogen to modulate virulence factor production in specific environments.

Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.

PubMed Central

Amselem, S; Sobrier, M L; Duquesnoy, P; Rappaport, R; Postel-Vinay, M C; Gourmelen, M; Dallapiccola, B; Goossens, M

1991-01-01

In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism. Images PMID:1999489

The genetics of early telencephalon patterning: some assembly required

PubMed Central

Hébert, Jean M.; Fishell, Gord

2009-01-01

The immense range of human behaviours is rooted in the complex neural networks of the cerebrum. The creation of these networks depends on the precise integration of specific neuronal subtypes that are born in different regions of the telencephalon. Here, using the mouse as a model system, we review how these proliferative zones are established. Moreover, we discuss how these regions can be traced back in development to the function of a few key genes, including those that encode fibroblast growth factors (FGFs), sonic hedgehog (SHH), bone morphogenetic proteins (BMPs), forkhead box G1 (FoxG1), paired box 6 (PAX6) and LIM homeobox protein 2 (LHX2), that pattern the early telencephalon. PMID:19143049

MET Receptor Tyrosine Kinase as an Autism Genetic Risk Factor

PubMed Central

Peng, Yun; Huentelman, Matthew; Smith, Christopher; Qiu, Shenfeng

2014-01-01

In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions. PMID:24290385

Content-Specific Source Encoding in the Human Medial Temporal Lobe

ERIC Educational Resources Information Center

Awipi, T.; Davachi, L.

2008-01-01

Although the medial temporal lobe (MTL) is known to be essential for episodic encoding, the contributions of individual MTL subregions remain unclear. Data from recognition memory studies have provided evidence that the hippocampus supports relational encoding important for later episodic recollection, whereas the perirhinal cortex has been linked…

Inhibition of poly(ADP-ribose) polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

PubMed

Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Schlesinger, Mariana; Venkannagari, Harikanth; Flawiá, Mirtha M; Villamil, Silvia H Fernández; Lehtiö, Lari

2012-01-01

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

Limited Dissemination of Extended-Spectrum β-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

PubMed

Börjesson, Stefan; Ny, Sofia; Egervärn, Maria; Bergström, Jakob; Rosengren, Åsa; Englund, Stina; Löfmark, Sonja; Byfors, Sara

2016-04-01

Extended-spectrum β-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.

On the Edge of Language Acquisition: Inherent Constraints on Encoding Multisyllabic Sequences in the Neonate Brain

ERIC Educational Resources Information Center

Ferry, Alissa L.; Fló, Ana; Brusini, Perrine; Cattarossi, Luigi; Macagno, Francesco; Nespor, Marina; Mehler, Jacques

2016-01-01

To understand language, humans must encode information from rapid, sequential streams of syllables--tracking their order and organizing them into words, phrases, and sentences. We used Near-Infrared Spectroscopy (NIRS) to determine whether human neonates are born with the capacity to track the positions of syllables in multisyllabic sequences.…

Basic anatomy and tumor biology of the RPS6KA6 gene that encodes the p90 ribosomal S6 kinase-4

PubMed Central

Sun, Yuan; Cao, Shousong; Yang, Min; Wu, Sihong; Wang, Zhe; Lin, Xiukun; Song, Xiangrang; Liao, D.J.

2012-01-01

The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5’RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon whereas two indolocarbazole-derived inhibitors of cyclin dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kD wild type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72-kD were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors. PMID:22614021

The prediction of biogenic magnetic nanoparticles biomineralization in human tissues and organs

NASA Astrophysics Data System (ADS)

Medviediev, O.; Gorobets, O. Yu; Gorobets, S. V.; Yadrykhins'ky, V. S.

2017-10-01

In this study, human homologs of magnetosome island proteins basing on pairwise and multiple alignment of amino acid sequences were found. The expression levels of genes, which encode magnetosome island proteins of M. gryphiswaldense MSR-1, that were cultured under oxygen deficiency conditions and also under microaerobic conditions were compared to the expression levels of genes that encode the relevant homologs in human organism. The possibility of BMN biomineralization in human tissues and organs, in which BMN were not experimentally found before, was predicted.

The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine.

PubMed

Roos, Viktoria; Ulett, Glen C; Schembri, Mark A; Klemm, Per

2006-01-01

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles of E. coli 83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular polysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABU E. coli and UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

NASA Technical Reports Server (NTRS)

Butel, J. S.

2000-01-01

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR

PubMed Central

Nishiyama, Shoko; Slack, Olga A. L.; Lokugamage, Nandadeva; Hill, Terence E.; Juelich, Terry L.; Zhang, Lihong; Smith, Jennifer K.; Perez, David; Gong, Bin; Freiberg, Alexander N.; Ikegami, Tetsuro

2016-01-01

ABSTRACT Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker. PMID:27248570

Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

PubMed

Nishiyama, Shoko; Slack, Olga A L; Lokugamage, Nandadeva; Hill, Terence E; Juelich, Terry L; Zhang, Lihong; Smith, Jennifer K; Perez, David; Gong, Bin; Freiberg, Alexander N; Ikegami, Tetsuro

2016-11-16

Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

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

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

Adenovirus-mediated E2-EPF UCP Gene Transfer Prevents Autoamputation in a Mouse Model of Hindlimb Ischemia

PubMed Central

Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

2012-01-01

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis. PMID:22294149

Adenovirus-mediated E2-EPF UCP gene transfer prevents autoamputation in a mouse model of hindlimb ischemia.

PubMed

Lim, Jung Hwa; Shin, Hyo Jung; Park, Kyeong-Su; Lee, Chan Hee; Jung, Cho-Rok; Im, Dong-Soo

2012-04-01

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

Cellodextrin Utilization by Bifidobacterium breve UCC2003▿ †

PubMed Central

Pokusaeva, Karina; O'Connell-Motherway, Mary; Zomer, Aldert; MacSharry, John; Fitzgerald, Gerald F.; van Sinderen, Douwe

2011-01-01

Cellodextrins, the incomplete hydrolysis products from insoluble cellulose, are accessible as a carbon source to certain members of the human gut microbiota, such as Bifidobacterium breve UCC2003. Transcription of the cldEFGC gene cluster of B. breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating this cluster in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this commensal. Moreover, our results suggest that transcription of the cld cluster is controlled by a LacI-type regulator encoded by cldR, located immediately upstream of cldE. Gel mobility shift assays using purified CldRHis (produced by the incorporation of a His12-encoding sequence into the 3′ end of the cldC gene) indicate that the cldEFGC promoter is subject to negative control by CldRHis, which binds to two inverted repeats. Analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose, and cellopentaose, with cellotriose apparently representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 is, to the best of our knowledge, the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity toward various cellodextrins. PMID:21216899

Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (Solanum lycopersicum L.)

PubMed Central

Humplík, Jan F.; Bergougnoux, Véronique; Jandová, Michaela; Šimura, Jan; Pěnčík, Aleš; Tomanec, Ondřej; Rolčík, Jakub; Novák, Ondřej; Fellner, Martin

2015-01-01

Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings. PMID:25695830

Deep--deeper--deepest? Encoding strategies and the recognition of human faces.

PubMed

Sporer, S L

1991-03-01

Various encoding strategies that supposedly promote deeper processing of human faces (e.g., character judgments) have led to better recognition than more shallow processing tasks (judging the width of the nose). However, does deeper processing actually lead to an improvement in recognition, or, conversely, does shallow processing lead to a deterioration in performance when compared with naturally employed encoding strategies? Three experiments systematically compared a total of 8 different encoding strategies manipulating depth of processing, amount of elaboration, and self-generation of judgmental categories. All strategies that required a scanning of the whole face were basically equivalent but no better than natural strategy controls. The consistently worst groups were the ones that rated faces along preselected physical dimensions. This can be explained by subjects' lesser task involvement as revealed by manipulation checks.

Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

PubMed Central

Buhren, Bettina Alexandra; Martinez, Cynthia; Schrumpf, Holger; Gasis, Marcia; Grether-Beck, Susanne; Krutmann, Jean

2013-01-01

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics. PMID:23840300

Cloning and characterization of cDNAs encoding human gastrin-releasing peptide.

PubMed Central

Spindel, E R; Chin, W W; Price, J; Rees, L H; Besser, G M; Habener, J F

1984-01-01

We have prepared and cloned cDNAs derived from poly(A)+ RNA from a human pulmonary carcinoid tumor rich in immunoreactivity to gastrin-releasing peptide, a peptide closely related in structure to amphibian bombesin. Mixtures of synthetic oligodeoxyribonucleotides corresponding to amphibian bombesin were used as hybridization probes to screen a cDNA library prepared from the tumor RNA. Sequencing of the recombinant plasmids shows that human gastrin-releasing peptide (hGRP) mRNA encodes a precursor of 148 amino acids containing a typical signal sequence, hGRP consisting of 27 or 28 amino acids, and a carboxyl-terminal extension peptide. hGRP is flanked at its carboxyl terminus by two basic amino acids, following a glycine used for amidation of the carboxyl-terminal methionine. RNA blot analyses of tumor RNA show a major mRNA of 900 bases and a minor mRNA of 850 bases. Blot hybridization analyses using human genomic DNA are consistent with a single hGRP-encoding gene. The presence of two mRNAs encoding the hGRP precursor protein in the face of a single hGRP gene raises the possibility of alternative processing of the single RNA transcript. Images PMID:6207529

ssrA (tmRNA) Plays a Role in Salmonella enterica Serovar Typhimurium Pathogenesis

PubMed Central

Julio, Steven M.; Heithoff, Douglas M.; Mahan, Michael J.

2000-01-01

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL, encoded within ssrA, serves as the site of integration of a region of Salmonella-specific sequence; adjacent to the 5′ end of ssrA is another region of Salmonella-specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella, serves as an attachment site for Salmonella-specific sequences, and is required for the growth of phage P22. PMID:10692360

SGRL can regulate chlorophyll metabolism and contributes to normal plant growth and development in Pisum sativum L.

PubMed

Bell, Andrew; Moreau, Carol; Chinoy, Catherine; Spanner, Rebecca; Dalmais, Marion; Le Signor, Christine; Bendahmane, Abdel; Klenell, Markus; Domoney, Claire

2015-12-01

Among a set of genes in pea (Pisum sativum L.) that were induced under drought-stress growth conditions, one encoded a protein with significant similarity to a regulator of chlorophyll catabolism, SGR. This gene, SGRL, is distinct from SGR in genomic location, encoded carboxy-terminal motif, and expression through plant and seed development. Divergence of the two encoded proteins is associated with a loss of similarity in intron/exon gene structure. Transient expression of SGRL in leaves of Nicotiana benthamiana promoted the degradation of chlorophyll, in a manner that was distinct from that shown by SGR. Removal of a predicted transmembrane domain from SGRL reduced its activity in transient expression assays, although variants with and without this domain reduced SGR-induced chlorophyll degradation, indicating that the effects of the two proteins are not additive. The combined data suggest that the function of SGRL during growth and development is in chlorophyll re-cycling, and its mode of action is distinct from that of SGR. Studies of pea sgrL mutants revealed that plants had significantly lower stature and yield, a likely consequence of reduced photosynthetic efficiencies in mutant compared with control plants under conditions of high light intensity.

Fatty acid-producing hosts

DOEpatents

Pfleger, Brian F; Lennen, Rebecca M

2013-12-31

Described are hosts for overproducing a fatty acid product such as a fatty acid. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the hosts are functionally delected. The hosts prefereably include the nucleic acid encoding the thioesterase at an intermediate copy number. The hosts are preferably recominantly stable and growth-competent at 37.degree. C. Methods of producing a fatty acid product comprising culturing such hosts at 37.degree. C. are also described.

Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

PubMed Central

Fisher, Susan H.; Wray, Lewis V.

2002-01-01

Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression. PMID:11914346

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

PubMed

Fisher, Susan H; Wray, Lewis V

2002-04-01

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

What Has Cancer Taught Us about the Cell?

ERIC Educational Resources Information Center

Hatton, Mary E.; Hatton, Mark P.

1997-01-01

Discusses what is cancer; proto-oncogenes that encode four classes of proteins including growth factors, growth factor receptors, intracellular signaling messengers, and transcription factors; tumor suppressors; and cancer therapy including metabolic inhibitors, alkylating agents and antibiotics, mitotic inhibitors, and hormone-related therapy.…

Deviance detection based on regularity encoding along the auditory hierarchy: electrophysiological evidence in humans.

PubMed

Escera, Carles; Leung, Sumie; Grimm, Sabine

2014-07-01

Detection of changes in the acoustic environment is critical for survival, as it prevents missing potentially relevant events outside the focus of attention. In humans, deviance detection based on acoustic regularity encoding has been associated with a brain response derived from the human EEG, the mismatch negativity (MMN) auditory evoked potential, peaking at about 100-200 ms from deviance onset. By its long latency and cerebral generators, the cortical nature of both the processes of regularity encoding and deviance detection has been assumed. Yet, intracellular, extracellular, single-unit and local-field potential recordings in rats and cats have shown much earlier (circa 20-30 ms) and hierarchically lower (primary auditory cortex, medial geniculate body, inferior colliculus) deviance-related responses. Here, we review the recent evidence obtained with the complex auditory brainstem response (cABR), the middle latency response (MLR) and magnetoencephalography (MEG) demonstrating that human auditory deviance detection based on regularity encoding-rather than on refractoriness-occurs at latencies and in neural networks comparable to those revealed in animals. Specifically, encoding of simple acoustic-feature regularities and detection of corresponding deviance, such as an infrequent change in frequency or location, occur in the latency range of the MLR, in separate auditory cortical regions from those generating the MMN, and even at the level of human auditory brainstem. In contrast, violations of more complex regularities, such as those defined by the alternation of two different tones or by feature conjunctions (i.e., frequency and location) fail to elicit MLR correlates but elicit sizable MMNs. Altogether, these findings support the emerging view that deviance detection is a basic principle of the functional organization of the auditory system, and that regularity encoding and deviance detection is organized in ascending levels of complexity along the auditory pathway expanding from the brainstem up to higher-order areas of the cerebral cortex.

Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer.

PubMed

Okada, Yoshiyuki; Sonoshita, Masahiro; Kakizaki, Fumihiko; Aoyama, Naoki; Itatani, Yoshiro; Uegaki, Masayuki; Sakamoto, Hiromasa; Kobayashi, Takashi; Inoue, Takahiro; Kamba, Tomomi; Suzuki, Akira; Ogawa, Osamu; Taketo, M Mark

2017-04-01

A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration-resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR-defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium-specific inactivation of Aes in Pten flox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Apoptotic role of TGF-β mediated by Smad4 mitochondria translocation and cytochrome c oxidase subunit II interaction.

PubMed

Pang, Lijuan; Qiu, Tao; Cao, Xu; Wan, Mei

2011-07-01

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones and plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic cells. Smad4 gene silencing by siRNA inhibits TGF-β-induced apoptosis in Hep3B cells and UV-induced apoptosis in PANC-1 cells. Cell fractionation assays demonstrated that a fraction of Smad4 translocates to mitochondria after long time TGF-β treatment or UV exposure, during which the cells were under apoptosis. Smad4 mitochondria translocation during apoptosis was also confirmed by fluorescence observation of Smad4 colocalization with MitoTracker Red. We searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, i.e., one clone encoded cytochrome c oxidase COXII, three clones encoded COXIII and one clone encoded COXVb. Strong interaction between Smad4 with COXII, an important apoptosis regulator, was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Further, mitochondrial portion of cells was isolated and the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure was confirmed. Importantly, targeting Smad4 to mitochondria using import leader fusions enhanced TGF-β-induced apoptosis. Collectively, the results suggest that Smad4 promote apoptosis of the cells through its mitochondrial translocation and association with mitochondria protein COXII. Copyright © 2011 Elsevier Inc. All rights reserved.

Glycoside hydrolase family 13 α-glucosidases encoded by Bifidobacterium breve UCC2003; A comparative analysis of function, structure and phylogeny.

PubMed

Kelly, Emer D; Bottacini, Francesca; O'Callaghan, John; Motherway, Mary O'Connell; O'Connell, Kerry Joan; Stanton, Catherine; van Sinderen, Douwe

2016-05-02

Bifidobacterium breve is a noted inhabitant and one of the first colonizers of the human gastro intestinal tract (GIT). The ability of this bacterium to persist in the GIT is reflected by the abundance of carbohydrate-active enzymes that are encoded by its genome. One such family of enzymes is represented by the α-glucosidases, of which three, Agl1, Agl2 and MelD, have previously been identified and characterized in the prototype B. breve strain UCC2003. In this report, we describe an additional B. breve UCC2003-encoded α-glucosidase, along with a B. breve UCC2003-encoded α-glucosidase-like protein, designated here as Agl3 and Agl4, respectively, which together with the three previously described enzymes belong to glycoside hydrolase (GH) family 13. Agl3 was shown to exhibit hydrolytic specificity towards the α-(1→6) linkage present in palatinose; the α-(1→3) linkage present in turanose; the α-(1→4) linkages found in maltotriose and maltose; and to a lesser degree, the α-(1→2) linkage found in sucrose and kojibiose; and the α-(1→5) linkage found in leucrose. Surprisingly, based on the substrates analyzed, Agl4 did not exhibit biologically relevant α-glucosidic activity. With the presence of four functionally active GH13 α-glucosidases, B. breve UCC2003 is capable of hydrolyzing all α-glucosidic linkages that can be expected in glycan substrates in the lower GIT. This abundance of α-glucosidases provides B. breve UCC2003 with an adaptive ability and metabolic versatility befitting the transient nature of growth substrates in the GIT. Copyright © 2016 Elsevier B.V. All rights reserved.

Transformed Neural Pattern Reinstatement during Episodic Memory Retrieval.

PubMed

Xiao, Xiaoqian; Dong, Qi; Gao, Jiahong; Men, Weiwei; Poldrack, Russell A; Xue, Gui

2017-03-15

Contemporary models of episodic memory posit that remembering involves the reenactment of encoding processes. Although encoding-retrieval similarity has been consistently reported and linked to memory success, the nature of neural pattern reinstatement is poorly understood. Using high-resolution fMRI on human subjects, our results obtained clear evidence for item-specific pattern reinstatement in the frontoparietal cortex, even when the encoding-retrieval pairs shared no perceptual similarity. No item-specific pattern reinstatement was found in the ventral visual cortex. Importantly, the brain regions and voxels carrying item-specific representation differed significantly between encoding and retrieval, and the item specificity for encoding-retrieval similarity was smaller than that for encoding or retrieval, suggesting different nature of representations between encoding and retrieval. Moreover, cross-region representational similarity analysis suggests that the encoded representation in the ventral visual cortex was reinstated in the frontoparietal cortex during retrieval. Together, these results suggest that, in addition to reinstatement of the originally encoded pattern in the brain regions that perform encoding processes, retrieval may also involve the reinstatement of a transformed representation of the encoded information. These results emphasize the constructive nature of memory retrieval that helps to serve important adaptive functions. SIGNIFICANCE STATEMENT Episodic memory enables humans to vividly reexperience past events, yet how this is achieved at the neural level is barely understood. A long-standing hypothesis posits that memory retrieval involves the faithful reinstatement of encoding-related activity. We tested this hypothesis by comparing the neural representations during encoding and retrieval. We found strong pattern reinstatement in the frontoparietal cortex, but not in the ventral visual cortex, that represents visual details. Critically, even within the same brain regions, the nature of representation during retrieval was qualitatively different from that during encoding. These results suggest that memory retrieval is not a faithful replay of past event but rather involves additional constructive processes to serve adaptive functions. Copyright © 2017 the authors 0270-6474/17/372986-13$15.00/0.

Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing.

PubMed

Fei, Teng; Chen, Yiwen; Xiao, Tengfei; Li, Wei; Cato, Laura; Zhang, Peng; Cotter, Maura B; Bowden, Michaela; Lis, Rosina T; Zhao, Shuang G; Wu, Qiu; Feng, Felix Y; Loda, Massimo; He, Housheng Hansen; Liu, X Shirley; Brown, Myles

2017-06-27

Alternative RNA splicing plays an important role in cancer. To determine which factors involved in RNA processing are essential in prostate cancer, we performed a genome-wide CRISPR/Cas9 knockout screen to identify the genes that are required for prostate cancer growth. Functional annotation defined a set of essential spliceosome and RNA binding protein (RBP) genes, including most notably heterogeneous nuclear ribonucleoprotein L (HNRNPL). We defined the HNRNPL-bound RNA landscape by RNA immunoprecipitation coupled with next-generation sequencing and linked these RBP-RNA interactions to changes in RNA processing. HNRNPL directly regulates the alternative splicing of a set of RNAs, including those encoding the androgen receptor, the key lineage-specific prostate cancer oncogene. HNRNPL also regulates circular RNA formation via back splicing. Importantly, both HNRNPL and its RNA targets are aberrantly expressed in human prostate tumors, supporting their clinical relevance. Collectively, our data reveal HNRNPL and its RNA clients as players in prostate cancer growth and potential therapeutic targets.

A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth

NASA Astrophysics Data System (ADS)

Holmgren, Lars; Ambrosino, Elena; Birot, Olivier; Tullus, Carl; Veitonmäki, Niina; Levchenko, Tetyana; Carlson, Lena-Maria; Musiani, Piero; Iezzi, Manuela; Curcio, Claudia; Forni, Guido; Cavallo, Federica; Kiessling, Rolf

2006-06-01

Endogenous angiogenesis inhibitors have shown promise in preclinical trials, but clinical use has been hindered by low half-life in circulation and high production costs. Here, we describe a strategy that targets the angiostatin receptor angiomotin (Amot) by DNA vaccination. The vaccination procedure generated antibodies that detected Amot on the endothelial cell surface. Purified Ig bound to the endothelial cell membrane and inhibited endothelial cell migration. In vivo, DNA vaccination blocked angiogenesis in the matrigel plug assay and prevented growth of transplanted tumors for up to 150 days. We further demonstrate that a combination of DNA vaccines encoding Amot and the extracellular and transmembrane domains of the human EGF receptor 2 (Her-2)/neu oncogene inhibited breast cancer progression and impaired tumor vascularization in Her-2/neu transgenic mice. No toxicity or impairment of normal blood vessels could be detected. This work shows that DNA vaccination targeting Amot may be used to mimic the effect of angiostatin. cancer vaccines | neoplasia | neovascularization | breast cancer | angiostatin

The Role of Neurotrophins in Major Depressive Disorder.

PubMed

Jiang, Cheng; Salton, Stephen R

2013-03-01

Neurotrophins and other growth factors have been advanced as critical modulators of depressive behavior. Support for this model is based on analyses of knockout and transgenic mouse models, human genetic studies, and screens for gene products that are regulated by depressive behavior and/or antidepressants. Even subtle alteration in the regulated secretion of brain-derived neurotrophic factor (BDNF), for example, due to a single nucleotide polymorphism (SNP)-encoded Val-Met substitution in proBDNF that affects processing and sorting, impacts behavior and cognition. Alterations in growth factor expression result in changes in neurogenesis as well as structural changes in neuronal cytoarchitecture, including effects on dendritic length and spine density, in the hippocampus, nucleus accumbens, and prefrontal cortex. These changes have the potential to impact the plasticity and stability of synapses in the CNS, and the complex brain circuitry that regulates behavior. Here we review the role that neurotrophins play in the modulation of depressive behavior, and the downstream signaling targets they regulate that potentially mediate these behavioral pro-depressant and antidepressant effects.

The Role of Neurotrophins in Major Depressive Disorder

PubMed Central

Jiang, Cheng; Salton, Stephen R.

2013-01-01

Neurotrophins and other growth factors have been advanced as critical modulators of depressive behavior. Support for this model is based on analyses of knockout and transgenic mouse models, human genetic studies, and screens for gene products that are regulated by depressive behavior and/or antidepressants. Even subtle alteration in the regulated secretion of brain-derived neurotrophic factor (BDNF), for example, due to a single nucleotide polymorphism (SNP)-encoded Val-Met substitution in proBDNF that affects processing and sorting, impacts behavior and cognition. Alterations in growth factor expression result in changes in neurogenesis as well as structural changes in neuronal cytoarchitecture, including effects on dendritic length and spine density, in the hippocampus, nucleus accumbens, and prefrontal cortex. These changes have the potential to impact the plasticity and stability of synapses in the CNS, and the complex brain circuitry that regulates behavior. Here we review the role that neurotrophins play in the modulation of depressive behavior, and the downstream signaling targets they regulate that potentially mediate these behavioral pro-depressant and antidepressant effects. PMID:23691270

Identification of apoptosis-related PLZF target genes

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

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

2007-07-27

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

An electrophysiological investigation of memory encoding, depth of processing, and word frequency in humans.

PubMed

Guo, Chunyan; Zhu, Ying; Ding, Jinhong; Fan, Silu; Paller, Ken A

2004-02-12

Memory encoding can be studied by monitoring brain activity correlated with subsequent remembering. To understand brain potentials associated with encoding, we compared multiple factors known to affect encoding. Depth of processing was manipulated by requiring subjects to detect animal names (deep encoding) or boldface (shallow encoding) in a series of Chinese words. Recognition was more accurate with deep than shallow encoding, and for low- compared to high-frequency words. Potentials were generally more positive for subsequently recognized versus forgotten words; for deep compared to shallow processing; and, for remembered words only, for low- than for high-frequency words. Latency and topographic differences between these potentials suggested that several factors influence the effectiveness of encoding and can be distinguished using these methods, even with Chinese logographic symbols.

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

PubMed Central

2011-01-01

Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants. PMID:21774824

Interrelations between Glycine Betaine Catabolism and Methionine Biosynthesis in Sinorhizobium meliloti Strain 102F34

PubMed Central

Barra, Lise; Fontenelle, Catherine; Ermel, Gwennola; Trautwetter, Annie; Walker, Graham C.; Blanco, Carlos

2006-01-01

Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B12-dependent enzyme is encoded by the metH gene. Glycine betaine can also serve as an alternative methyl donor for homocysteine. This reaction is catalyzed by betaine-homocysteine methyl transferase (BHMT), an enzyme that has been characterized in humans and rats. An S. meliloti gene whose product is related to the human BHMT enzyme has been identified and named bmt. This enzyme is closely related to mammalian BHMTs but has no homology with previously described bacterial betaine methyl transferases. Glycine betaine inhibits the growth of an S. meliloti bmt mutant in low- and high-osmotic strength media, an effect that correlates with a decrease in the catabolism of glycine betaine. This inhibition was not observed with other betaines, like homobetaine, dimethylsulfoniopropionate, and trigonelline. The addition of methionine to the growth medium allowed a bmt mutant to recover growth despite the presence of glycine betaine. Methionine also stimulated glycine betaine catabolism in a bmt strain, suggesting the existence of another catabolic pathway. Inactivation of metH or bmt did not affect the nodulation efficiency of the mutants in the 102F34 strain background. Nevertheless, a metH strain was severely defective in competing with the wild-type strain in a coinoculation experiment. PMID:17015658

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

PubMed

Tanaka, Motonari; Nanba, Daisuke; Mori, Seiji; Shiba, Fumio; Ishiguro, Hiroshi; Yoshino, Koichiro; Matsuura, Nariaki; Higashiyama, Shigeki

2004-10-01

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

Gene ercA, encoding a putative iron-containing alcohol dehydrogenase, is involved in regulation of ethanol utilization in Pseudomonas aeruginosa.

PubMed

Hempel, Niels; Görisch, Helmut; Mern, Demissew S

2013-09-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported.

Gene ercA, Encoding a Putative Iron-Containing Alcohol Dehydrogenase, Is Involved in Regulation of Ethanol Utilization in Pseudomonas aeruginosa

PubMed Central

Hempel, Niels; Görisch, Helmut

2013-01-01

Several two-component regulatory systems are known to be involved in the signal transduction pathway of the ethanol oxidation system in Pseudomonas aeruginosa ATCC 17933. These sensor kinases and response regulators are organized in a hierarchical manner. In addition, a cytoplasmic putative iron-containing alcohol dehydrogenase (Fe-ADH) encoded by ercA (PA1991) has been identified to play an essential role in this regulatory network. The gene ercA (PA1991) is located next to ercS, which encodes a sensor kinase. Inactivation of ercA (PA1991) by insertion of a kanamycin resistance cassette created mutant NH1. NH1 showed poor growth on various alcohols. On ethanol, NH1 grew only with an extremely extended lag phase. During the induction period on ethanol, transcription of structural genes exa and pqqABCDEH, encoding components of initial ethanol oxidation in P. aeruginosa, was drastically reduced in NH1, which indicates the regulatory function of ercA (PA1991). However, transcription in the extremely delayed logarithmic growth phase was comparable to that in the wild type. To date, the involvement of an Fe-ADH in signal transduction processes has not been reported. PMID:23813731

Cloning and characterization of largemouth bass ( Micropterus salmoides) myostatin encoding gene and its promoter

NASA Astrophysics Data System (ADS)

Li, Shengjie; Bai, Junjie; Wang, Lin

2008-08-01

Myostatin or GDF-8, a member of the transforming growth factor-β (TGF-β) superfamily, has been demonstrated to be a negative regulator of skeletal muscle mass in mammals. In the present study, we obtained a 5.64 kb sequence of myostatin encoding gene and its promoter from largemouth bass ( Micropterus salmoides). The myostatin encoding gene consisted of three exons (488 bp, 371 bp and 1779 bp, respectively) and two introns (390 bp and 855 bp, respectively). The intron-exon boundaries were conservative in comparison with those of mammalian myostatin encoding genes, whereas the size of introns was smaller than that of mammals. Sequence analysis of 1.569 kb of the largemouth bass myostatin gene promoter region revealed that it contained two TATA boxes, one CAAT box and nine putative E-boxes. Putative muscle growth response elements for myocyte enhancer factor 2 (MEF2), serum response factor (SRF), activator protein 1 (AP1), etc., and muscle-specific Mt binding site (MTBF) were also detected. Some of the transcription factor binding sites were conserved among five teleost species. This information will be useful for studying the transcriptional regulation of myostatin in fish.

Characterization of interleukin-8 receptors in non-human primates

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

Alvarez, V.; Coto, E.; Gonzalez-Roces, S.

Interleukin-8 is a chemokine with a potent neutrophil chemoatractant activity. In humans, two different cDNAs encoding human IL8 receptors designated IL8RA and IL8RB have been cloned. IL8RA binds IL8, while IL8RB binds IL8 as well as other {alpha}-chemokines. Both human IL8Rs are encoded by two genes physically linked on chromosome 2. The IL8RA and IL8RB genes have open reading frames (ORF) lacking introns. By direct sequencing of the polymerase chain reaction products, we sequenced the IL8R genes of cell lines from four non-human primates: chimpanzee, gorilla, orangutan, and macaca. The IL8RB encodes an ORF in the four non-human primates, showingmore » 95%-99% similarity to the human IL8RB sequence. The IL8RA homologue in gorilla and chimpanzee consisted of two ORF 98%-99% identical to the human sequence. The macaca and orangutan IL8RA homologues are pseudogenes: a 2 base pair insertion generated a sequence with several stop codons. In addition, we describe the physical linkage of these genes in the four non-human primates and discuss the evolutionary implications of these findings. 25 refs., 5 figs., 3 tabs.« less

RBM28, a protein deficient in ANE syndrome, regulates hair follicle growth via miR-203 and p63.

PubMed

Warshauer, Emily; Samuelov, Liat; Sarig, Ofer; Vodo, Dan; Bindereif, Albrecht; Kanaan, Moien; Gat, Uri; Fuchs-Telem, Dana; Shomron, Noam; Farberov, Luba; Pasmanik-Chor, Metsada; Nardini, Gil; Winkler, Eyal; Meilik, Benjamin; Petit, Isabelle; Aberdam, Daniel; Paus, Ralf; Sprecher, Eli; Nousbeck, Janna

2015-08-01

Alopecia-neurological defects-endocrinopathy (ANE) syndrome is a rare inherited hair disorder, which was shown to result from decreased expression of the RNA-binding motif protein 28 (RBM28). In this study, we attempted to delineate the role of RBM28 in hair biology. First, we sought to obtain evidence for the direct involvement of RBM28 in hair growth. When RBM28 was downregulated in human hair follicle (HF) organ cultures, we observed catagen induction and HF growth arrest, indicating that RBM28 is necessary for normal hair growth. We also aimed at identifying molecular targets of RBM28. Given that an RBM28 homologue was recently found to regulate miRNA biogenesis in C. elegans and given the known pivotal importance of miRNAs for proper hair follicle development, we studied global miRNA expression profile in cells knocked down for RBM28. This analysis revealed that RBM28 controls the expression of miR-203. miR-203 was found to regulate in turn TP63, encoding the transcription factor p63, which is critical for hair morphogenesis. In conclusion, RBM28 contributes to HF growth regulation through modulation of miR-203 and p63 activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

On the edge of language acquisition: inherent constraints on encoding multisyllabic sequences in the neonate brain.

PubMed

Ferry, Alissa L; Fló, Ana; Brusini, Perrine; Cattarossi, Luigi; Macagno, Francesco; Nespor, Marina; Mehler, Jacques

2016-05-01

To understand language, humans must encode information from rapid, sequential streams of syllables - tracking their order and organizing them into words, phrases, and sentences. We used Near-Infrared Spectroscopy (NIRS) to determine whether human neonates are born with the capacity to track the positions of syllables in multisyllabic sequences. After familiarization with a six-syllable sequence, the neonate brain responded to the change (as shown by an increase in oxy-hemoglobin) when the two edge syllables switched positions but not when two middle syllables switched positions (Experiment 1), indicating that they encoded the syllables at the edges of sequences better than those in the middle. Moreover, when a 25 ms pause was inserted between the middle syllables as a segmentation cue, neonates' brains were sensitive to the change (Experiment 2), indicating that subtle cues in speech can signal a boundary, with enhanced encoding of the syllables located at the edges of that boundary. These findings suggest that neonates' brains can encode information from multisyllabic sequences and that this encoding is constrained. Moreover, subtle segmentation cues in a sequence of syllables provide a mechanism with which to accurately encode positional information from longer sequences. Tracking the order of syllables is necessary to understand language and our results suggest that the foundations for this encoding are present at birth. © 2015 John Wiley & Sons Ltd.

Functional metagenomics to mine the human gut microbiome for dietary fiber catabolic enzymes.

PubMed

Tasse, Lena; Bercovici, Juliette; Pizzut-Serin, Sandra; Robe, Patrick; Tap, Julien; Klopp, Christophe; Cantarel, Brandi L; Coutinho, Pedro M; Henrissat, Bernard; Leclerc, Marion; Doré, Joël; Monsan, Pierre; Remaud-Simeon, Magali; Potocki-Veronese, Gabrielle

2010-11-01

The human gut microbiome is a complex ecosystem composed mainly of uncultured bacteria. It plays an essential role in the catabolism of dietary fibers, the part of plant material in our diet that is not metabolized in the upper digestive tract, because the human genome does not encode adequate carbohydrate active enzymes (CAZymes). We describe a multi-step functionally based approach to guide the in-depth pyrosequencing of specific regions of the human gut metagenome encoding the CAZymes involved in dietary fiber breakdown. High-throughput functional screens were first applied to a library covering 5.4 × 10(9) bp of metagenomic DNA, allowing the isolation of 310 clones showing beta-glucanase, hemicellulase, galactanase, amylase, or pectinase activities. Based on the results of refined secondary screens, sequencing efforts were reduced to 0.84 Mb of nonredundant metagenomic DNA, corresponding to 26 clones that were particularly efficient for the degradation of raw plant polysaccharides. Seventy-three CAZymes from 35 different families were discovered. This corresponds to a fivefold target-gene enrichment compared to random sequencing of the human gut metagenome. Thirty-three of these CAZy encoding genes are highly homologous to prevalent genes found in the gut microbiome of at least 20 individuals for whose metagenomic data are available. Moreover, 18 multigenic clusters encoding complementary enzyme activities for plant cell wall degradation were also identified. Gene taxonomic assignment is consistent with horizontal gene transfer events in dominant gut species and provides new insights into the human gut functional trophic chain.

When encoding yields remembering: insights from event-related neuroimaging.

PubMed Central

Wagner, A D; Koutstaal, W; Schacter, D L

1999-01-01

To understand human memory, it is important to determine why some experiences are remembered whereas others are forgotten. Until recently, insights into the neural bases of human memory encoding, the processes by which information is transformed into an enduring memory trace, have primarily been derived from neuropsychological studies of humans with select brain lesions. The advent of functional neuroimaging methods, such as positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), has provided a new opportunity to gain additional understanding of how the brain supports memory formation. Importantly, the recent development of event-related fMRI methods now allows for examination of trial-by-trial differences in neural activity during encoding and of the consequences of these differences for later remembering. In this review, we consider the contributions of PET and fMRI studies to the understanding of memory encoding, placing a particular emphasis on recent event-related fMRI studies of the Dm effect: that is, differences in neural activity during encoding that are related to differences in subsequent memory. We then turn our attention to the rich literature on the Dm effect that has emerged from studies using event-related potentials (ERPs). It is hoped that the integration of findings from ERP studies, which offer higher temporal resolution, with those from event-related fMRI studies, which offer higher spatial resolution, will shed new light on when and why encoding yields subsequent remembering. PMID:10466153

The use of enzymopathic human red cells in the study of malarial parasite glucose metabolism.

PubMed

Roth, E; Joulin, V; Miwa, S; Yoshida, A; Akatsuka, J; Cohen-Solal, M; Rosa, R

1988-05-01

The in vitro growth of Plasmodium falciparum malaria parasites was assayed in mutant red cells deficient in either diphosphoglycerate mutase (DPGM) or phosphoglycerate kinase (PGK). In addition, cDNA probes developed for human DNA sequences coding for these enzymes were used to examine the parasite genome by means of restriction endonuclease digestion and Southern blot analysis of parasite DNA. In both types of enzymopathic red cells, parasite growth was normal. In infected DPGM deficient red cells, no DPGM activity could be detected, and in normal red cells, DPGM activity declined slightly in a manner suggestive of parasite catabolism of host protein. However, in infected PGK deficient red cells, there was a 100-fold increase in PGK activity, and in normal red cells, a threefold increase in PGK activity was observed. Parasite PGK could be recovered from isolated parasites, and a marked increase in heat instability of parasite PGK as compared with the host cell enzyme was noted. Neither cDNA probe was found to cross-react with DNA sequences in the parasite genome. It is concluded that the parasite has no requirement for DPGM, and probably has no gene for this enzyme. On the other hand, the parasite does require PGK, (an adenosine triphosphate [ATP] generating enzyme) and synthesizes its own enzyme, which must have been encoded in the parasite genome. The parasite PGK gene most likely lacks sufficient homology to be detected by a human cDNA probe. Enzymopathic red cells are useful tools for elucidating the glycolytic enzymology of parasites and their co-evolution with their human hosts.

The 5-HT{sub 2A} serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

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

Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.

2006-05-19

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

PubMed

Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

2015-02-18

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). Copyright © 2015, American Association for the Advancement of Science.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

PubMed

Rivera, Frances E; Miller, Halie K; Kolar, Stacey L; Stevens, Stanley M; Shaw, Lindsey N

2012-01-01

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional Analysis of d-Alanylation of Lipoteichoic Acid in the Probiotic Strain Lactobacillus rhamnosus GG▿

PubMed Central

Vélez, Mónica Perea; Verhoeven, Tine L. A.; Draing, Christian; Von Aulock, Sonja; Pfitzenmaier, Markus; Geyer, Armin; Lambrichts, Ivo; Grangette, Corinne; Pot, Bruno; Vanderleyden, Jos; De Keersmaecker, Sigrid C. J.

2007-01-01

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. d-Alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of d-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results. PMID:17434999

G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

PubMed

Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

2015-01-01

Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Human parainfluenza virus type 2 V protein inhibits caspase-1.

PubMed

Ohta, Keisuke; Matsumoto, Yusuke; Nishio, Machiko

2018-04-01

The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1β (IL-1β) in a dose-dependent manner. IL-1β secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1β via its interaction with caspase-1.

Motivated encoding selectively promotes memory for future inconsequential semantically-related events.

PubMed

Oyarzún, Javiera P; Packard, Pau A; de Diego-Balaguer, Ruth; Fuentemilla, Lluis

2016-09-01

Neurobiological models of long-term memory explain how memory for inconsequential events fades, unless these happen before or after other relevant (i.e., rewarding or aversive) or novel events. Recently, it has been shown in humans that retrospective and prospective memories are selectively enhanced if semantically related events are paired with aversive stimuli. However, it remains unclear whether motivating stimuli, as opposed to aversive, have the same effect in humans. Here, participants performed a three phase incidental encoding task where one semantic category was rewarded during the second phase. A memory test 24h after, but not immediately after encoding, revealed that memory for inconsequential items was selectively enhanced only if items from the same category had been previously, but not subsequently, paired with rewards. This result suggests that prospective memory enhancement of reward-related information requires, like previously reported for aversive memories, of a period of memory consolidation. The current findings provide the first empirical evidence in humans that the effects of motivated encoding are selectively and prospectively prolonged over time. Copyright © 2016 Elsevier Inc. All rights reserved.

Nucleic acids encoding human trithorax protein

DOEpatents

Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

2001-01-01

In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

Oil Body-Bound Oleosin-rhFGF-10: A Novel Drug Delivery System that Improves Skin Penetration to Accelerate Wound Healing and Hair Growth in Mice

PubMed Central

Yang, Jing; Cai, Jingbo; Wang, Hongyu; Tian, Haishan; Huang, Jian; Qiang, Weidong; Zhang, Linbo; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-01-01

Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth. PMID:29057820

Oil Body-Bound Oleosin-rhFGF-10: A Novel Drug Delivery System that Improves Skin Penetration to Accelerate Wound Healing and Hair Growth in Mice.

PubMed

Li, Wenqing; Yang, Jing; Cai, Jingbo; Wang, Hongyu; Tian, Haishan; Huang, Jian; Qiang, Weidong; Zhang, Linbo; Li, Haiyan; Li, Xiaokun; Jiang, Chao

2017-10-18

Recombinant human fibroblast growth factor 10 (rhFGF-10) is frequently used to treat patients with skin injuries. It can also promote hair growth. However, the effective application of rhFGF-10 is limited because of its poor stability and transdermal absorption. In this study, polymerase chain reaction (PCR) and Southern blotting were used to identify transgenic safflowers carrying a gene encoding an oleosin-rhFGF-10 fusion protein. The size and structural integrity of oleosin-rhFGF-10 in oil bodies extracted from transgenic safflower seeds was characterized by polyacrylamide gel electrophoresis and western blotting. Oil body extracts containing oleosin-rhFGF-10 were topically applied to mouse skin. The absorption of oleosin-rhFGF-10 was studied by immunohistochemistry. Its efficiency in promoting wound healing and hair regeneration were evaluated in full thickness wounds and hair growth assays. We identified a safflower line that carried the transgene and expressed a 45 kDa oleosin-rhFGF-10 protein. Oil body-bound oleosin-rhFGF-10 was absorbed by the skin with higher efficiency and speed compared with prokaryotically-expressed rhFGF-10. Oleosin-rhFGF-10 also enhanced wound closure and promoted hair growth better than rhFGF-10. The application of oleosin-rhFGF-10 in oil bodies promoted its delivery through the skin, providing a basis for improved therapeutic effects in enhancing wound healing and hair growth.

Parallel Mutations Result in a Wide Range of Cooperation and Community Consequences in a Two-Species Bacterial Consortium

DOE PAGES

Douglas, Sarah M.; Chubiz, Lon M.; Harcombe, William R.; ...

2016-09-12

Multi-species microbial communities play a critical role in human health, industry, and waste remediation. Recently, the evolution of synthetic consortia in the laboratory has enabled adaptation to be addressed in the context of interacting species. Using an engineered bacterial consortium,we repeatedly evolved cooperative genotypes and examined both the predictability of evolution and the phenotypes that determinecommunity dynamics. Eight Salmonella enterica serovar Typhimurium strains evolved methionine excretion sufficient to support growth of an Escherichia coli methionine auxotroph, from whom they required excreted growth substrates. Non-synonymousmutations in metA, encoding homoserine trans-succinylase (HTS), were detected in each evolved S. enterica methionine cooperator andmore » were shown to be necessary for cooperative consortia growth. Molecular modeling was used to predict that most of the non-synonymous mutations slightly increase the binding affinity for HTS homodimer formation. Despite this genetic parallelism and trend of increasing protein binding stability, these metA alleles gave rise to a wide range of phenotypic diversity in termsof individual versus group benefit. The cooperators with the highest methionine excretion permitted nearly two-fold faster consortia growth and supported the highest fraction of E. coli, yet also had the slowest individual growth rates compared to less cooperative strains. Thus, although the genetic basis of adaptation was quite similar across independent origins of cooperative phenotypes, quantitative measurements of metabolite production were required to predict either the individual-level growth consequences or how these propagate to community-level behavior.« less

Parallel Mutations Result in a Wide Range of Cooperation and Community Consequences in a Two-Species Bacterial Consortium

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

Douglas, Sarah M.; Chubiz, Lon M.; Harcombe, William R.

Multi-species microbial communities play a critical role in human health, industry, and waste remediation. Recently, the evolution of synthetic consortia in the laboratory has enabled adaptation to be addressed in the context of interacting species. Using an engineered bacterial consortium,we repeatedly evolved cooperative genotypes and examined both the predictability of evolution and the phenotypes that determinecommunity dynamics. Eight Salmonella enterica serovar Typhimurium strains evolved methionine excretion sufficient to support growth of an Escherichia coli methionine auxotroph, from whom they required excreted growth substrates. Non-synonymousmutations in metA, encoding homoserine trans-succinylase (HTS), were detected in each evolved S. enterica methionine cooperator andmore » were shown to be necessary for cooperative consortia growth. Molecular modeling was used to predict that most of the non-synonymous mutations slightly increase the binding affinity for HTS homodimer formation. Despite this genetic parallelism and trend of increasing protein binding stability, these metA alleles gave rise to a wide range of phenotypic diversity in termsof individual versus group benefit. The cooperators with the highest methionine excretion permitted nearly two-fold faster consortia growth and supported the highest fraction of E. coli, yet also had the slowest individual growth rates compared to less cooperative strains. Thus, although the genetic basis of adaptation was quite similar across independent origins of cooperative phenotypes, quantitative measurements of metabolite production were required to predict either the individual-level growth consequences or how these propagate to community-level behavior.« less

MET receptor tyrosine kinase as an autism genetic risk factor.

PubMed

Peng, Yun; Huentelman, Matthew; Smith, Christopher; Qiu, Shenfeng

2013-01-01

In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions. © 2013 Elsevier Inc. All rights reserved.

Oncogenes in retroviruses and cells

NASA Astrophysics Data System (ADS)

Kurth, Reinhard

1983-09-01

Oncogenes are genes that cause cancer. Retroviruses contain oncogenes and cause cancer in animals and, perhaps, in man. The viruses have appropriated their oncogenes from normal cellular DNA by genetic recombination. Correspondingly, uninfected vertebrate cells contain a family of evolutionary conserved cellular oncogenes. Retrovirus infection, introducing additional viral oncogenes into the cells, as well as carcinogen-mediated activation of cellular oncogenes may both lead to increased synthesis of oncogene encoded transforming proteins which convert normal cells to tumor cells. Unique retroviruses of human origin have recently been identified. They may, on occasion, directly cause tumors in man. However, the general significance of retroviruses may better be illustrated by their remarkable genetic composition which allows them to promote tumor growth by a variety of genetic mechanisms.

Emerging Functions for the Staphylococcus aureus RNome

PubMed Central

Felden, Brice

2013-01-01

Staphylococcus aureus is a leading pathogen for animals and humans, not only being one of the most frequently isolated bacteria in hospital-associated infections but also causing diseases in the community. To coordinate the expression of its numerous virulence genes for growth and survival, S. aureus uses various signalling pathways that include two-component regulatory systems, transcription factors, and also around 250 regulatory RNAs. Biological roles have only been determined for a handful of these sRNAs, including cis, trans, and cis-trans acting RNAs, some internally encoding small, functional peptides and others possessing dual or multiple functions. Here we put forward an inventory of these fascinating sRNAs; the proteins involved in their activities; and those involved in stress response, metabolisms, and virulence. PMID:24348246

Yersinia Type III Secretion System Master Regulator LcrF

PubMed Central

Schwiesow, Leah; Lam, Hanh

2015-01-01

Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. PMID:26644429

Localization and physical mapping of genes encoding the A+U-rich element RNA-binding protein AUF1 to human chromosomes 4 and X

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

Wagner, B.J.; Long, L.; Pettenati, M.J.

Messenger RNAs encoding many oncoproteins and cytokines are relatively unstable. Their instability, which ensures appropriate levels and timing of expression, is controlled in part by proteins that bind to A + U-rich instability elements (AREs) present in the 3{prime}-untranslated regions of the mRNAs. cDNAs encoding the AUF1 family of ARE-binding proteins were cloned from human and murine cDNA libraries. In the present study monochromosomal somatic cell hybrids were used to localize two AUF1 loci to human chromosomes 4 and X. In situ hybridization analyses using P1 clones as probes identified the 4q21.1-q21.2 and Xq12 regions as the locations of themore » AUF1 genes. 10 refs., 2 figs.« less

Concurrent encoding of frequency and amplitude modulation in human auditory cortex: encoding transition.

PubMed

Luo, Huan; Wang, Yadong; Poeppel, David; Simon, Jonathan Z

2007-12-01

Complex natural sounds (e.g., animal vocalizations or speech) can be characterized by specific spectrotemporal patterns the components of which change in both frequency (FM) and amplitude (AM). The neural coding of AM and FM has been widely studied in humans and animals but typically with either pure AM or pure FM stimuli. The neural mechanisms employed to perceptually unify AM and FM acoustic features remain unclear. Using stimuli with simultaneous sinusoidal AM (at rate f(AM) = 37 Hz) and FM (with varying rates f(FM)), magnetoencephalography (MEG) is used to investigate the elicited auditory steady-state response (aSSR) at relevant frequencies (f(AM), f(FM), f(AM) + f(FM)). Previous work demonstrated that for sounds with slower FM dynamics (f(FM) < 5 Hz), the phase of the aSSR at f(AM) tracked the FM; in other words, AM and FM features were co-tracked and co-represented by "phase modulation" encoding. This study explores the neural coding mechanism for stimuli with faster FM dynamics (< or =30 Hz), demonstrating that at faster rates (f(FM) > 5 Hz), there is a transition from pure phase modulation encoding to a single-upper-sideband (SSB) response (at frequency f(AM) + f(FM)) pattern. We propose that this unexpected SSB response can be explained by the additional involvement of subsidiary AM encoding responses simultaneously to, and in quadrature with, the ongoing phase modulation. These results, using MEG to reveal a possible neural encoding of specific acoustic properties, demonstrate more generally that physiological tests of encoding hypotheses can be performed noninvasively on human subjects, complementing invasive, single-unit recordings in animals.

Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver.

PubMed

Yu, Ping; Liu, Zhiquan; Wu, Donglei; Chen, Minghai; Lv, Weiwei; Zhao, Yunlong

2018-07-01

As a widespread and ubiquitous pollutant of marine ecosystems, microplastic has the potential to become an emerging global threat for aquatic organisms. The present study aims to elucidate the effects of microplastics on the growth, accumulation and oxidative stress response in the liver of Eriocheir sinensis. Fluorescent microplastic particles (diameter = 0.5 μm) accumulated in the gill, liver and gut tissues of E. sinensis were investigated when crabs were exposed to a concentration of 40000 μg/L for 7 days. A 21 day toxicity test suggested that the rate of weight gain, specific growth rate, and hepatosomatic index of E. sinensis decreased with increasing microplastic concentration (0 μg/L, 40 μg/L, 400 μg/L, 4000 μg/L and 40000 μg/L). The activities of AChE and GPT in crabs exposed to microplastics were lower than those in control group. GOT activity increased significantly after exposure to a low concentration of microplastics and then decreased continuously with increasing microplastic concentrations. The activities of superoxide dismutase (SOD), aspartate transaminase (GOT), glutathione (GSH), and glutathione peroxidase (GPx) increased in specimens exposed to low concentrations of microplastics (40 and 400 μg/L) compared to the control and decreased in organisms exposed to high concentrations (4000 and 40000 μg/L). In contrast, the activities of acetylcholinesterase, catalase (CAT), and alanine aminotransferase were significantly lower in the organisms exposed to microplastics compared to control animals. Upon exposure to increasing microplastic concentrations, the expression of genes encoding the antioxidants SOD, CAT, GPx and glutathione S-transferase in the liver decreased after first increasing. Exposure to microplastics increased the expression of the gene encoding p38 in the MAPK signaling pathway and significantly decreased the expressions of genes encoding ERK, AKT, and MEK. The results of this study demonstrate that microplastics can accumulate in the tissues of E. sinensis and negatively affect growth. In addition, exposure to microplastics causes damage and induces oxidative stress in the hepatopancreas of E. sinensis. The findings provide basic biological data for environmental and human risk assessments of microplastics of high concern. Copyright © 2018 Elsevier B.V. All rights reserved.

The Staphylococcus aureus leucine aminopeptidase LAP is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine

PubMed Central

Carroll, Ronan K.; Veillard, Florian; Gagne, Danielle T.; Lindenmuth, Jarrod M.; Poreba, Marcin; Drag, Marcin; Potempa, Jan; Shaw, Lindsey N.

2013-01-01

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part, due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase - LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria, however, in systemic and localized infection models the pepZ mutant was significantly attenuated in virulence. Recently, a contradictory report has been published, suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition we conduct a biochemical investigation of purified recombinant LAP identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine and that leucine is not the primary target of LAP. PMID:23241672

Resurrecting KIR2DP1: A Key Intermediate in the Evolution of Human Inhibitory NK Cell Receptors That Recognize HLA-C.

PubMed

Hilton, Hugo G; Blokhuis, Jeroen H; Guethlein, Lisbeth A; Norman, Paul J; Parham, Peter

2017-03-01

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1 F , the functional antecedent of KIR2DP1 We demonstrate how K44-KIR2DP1 F with lysine 44 recognized C1 + HLA-C, whereas T44-KIR2DP1 F recognized C2 + HLA-C. Dimorphisms at 12 other KIR2DP1 F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1 , KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA , C1-specific K44-KIR2DP1 F were stronger receptors than the attenuated C2-specific T44-KIR2DP1 F encoded on CenB The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR , likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1 F has properties consistent with KIR2DP1 F having been the founder gene. The first KIR2DP1 F alleles encoded K44-C1 receptors; subsequently KIR2DP1 F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1 F Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1 F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1 F by micro gene conversion. Copyright © 2017 by The American Association of Immunologists, Inc.

The Epstein-Barr Virus BART miRNA Cluster of the M81 Strain Modulates Multiple Functions in Primary B Cells

PubMed Central

Lin, Xiaochen; Tsai, Ming-Han; Shumilov, Anatoliy; Poirey, Remy; Bannert, Helmut; Middeldorp, Jaap M.; Feederle, Regina; Delecluse, Henri-Jacques

2015-01-01

The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects the majority of the human population. All EBV strains transform B lymphocytes, but some strains, such as M81, also induce spontaneous virus replication. EBV encodes 22 microRNAs (miRNAs) that form a cluster within the BART region of the virus and have been previously been found to stimulate tumor cell growth. Here we describe their functions in B cells infected by M81. We found that the BART miRNAs are downregulated in replicating cells, and that exposure of B cells in vitro or in vivo in humanized mice to a BART miRNA knockout virus resulted in an increased proportion of spontaneously replicating cells, relative to wild type virus. The BART miRNAs subcluster 1, and to a lesser extent subcluster 2, prevented expression of BZLF1, the key protein for initiation of lytic replication. Thus, multiple BART miRNAs cooperate to repress lytic replication. The BART miRNAs also downregulated pro- and anti-apoptotic mediators such as caspase 3 and LMP1, and their deletion did not sensitize B-cells to apoptosis. To the contrary, the majority of humanized mice infected with the BART miRNA knockout mutant developed tumors more rapidly, probably due to enhanced LMP1 expression, although deletion of the BART miRNAs did not modify the virus transforming abilities in vitro. This ability to slow cell growth could be confirmed in non-humanized immunocompromized mice. Injection of resting B cells exposed to a virus that lacks the BART miRNAs resulted in accelerated tumor growth, relative to wild type controls. Therefore, we found that the M81 BART miRNAs do not enhance B-cell tumorigenesis but rather repress it. The repressive effects of the BART miRNAs on potentially pathogenic viral functions in infected B cells are likely to facilitate long-term persistence of the virus in the infected host. PMID:26694854

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods

PubMed Central

Mulindwa, Julius; Leiss, Kevin; Ibberson, David; Kamanyi Marucha, Kevin; Helbig, Claudia; Melo do Nascimento, Larissa; Silvester, Eleanor; Matthews, Keith; Matovu, Enock; Enyaru, John

2018-01-01

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs. PMID:29474390

Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods.

PubMed

Mulindwa, Julius; Leiss, Kevin; Ibberson, David; Kamanyi Marucha, Kevin; Helbig, Claudia; Melo do Nascimento, Larissa; Silvester, Eleanor; Matthews, Keith; Matovu, Enock; Enyaru, John; Clayton, Christine

2018-02-01

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs.

JPEG2000 encoding with perceptual distortion control.

PubMed

Liu, Zhen; Karam, Lina J; Watson, Andrew B

2006-07-01

In this paper, a new encoding approach is proposed to control the JPEG2000 encoding in order to reach a desired perceptual quality. The new method is based on a vision model that incorporates various masking effects of human visual perception and a perceptual distortion metric that takes spatial and spectral summation of individual quantization errors into account. Compared with the conventional rate-based distortion minimization JPEG2000 encoding, the new method provides a way to generate consistent quality images at a lower bit rate.

Detection of the human endogenous retrovirus ERV3-encoded Env-protein in human tissues using antibody-based proteomics.

PubMed

Fei, Chen; Atterby, Christina; Edqvist, Per-Henrik; Pontén, Fredrik; Zhang, Wei Wei; Larsson, Erik; Ryan, Frank P

2014-01-01

There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to human evolution, physiology and disease.

The ENCODE Project at UC Santa Cruz.

PubMed

Thomas, Daryl J; Rosenbloom, Kate R; Clawson, Hiram; Hinrichs, Angie S; Trumbower, Heather; Raney, Brian J; Karolchik, Donna; Barber, Galt P; Harte, Rachel A; Hillman-Jackson, Jennifer; Kuhn, Robert M; Rhead, Brooke L; Smith, Kayla E; Thakkapallayil, Archana; Zweig, Ann S; Haussler, David; Kent, W James

2007-01-01

The goal of the Encyclopedia Of DNA Elements (ENCODE) Project is to identify all functional elements in the human genome. The pilot phase is for comparison of existing methods and for the development of new methods to rigorously analyze a defined 1% of the human genome sequence. Experimental datasets are focused on the origin of replication, DNase I hypersensitivity, chromatin immunoprecipitation, promoter function, gene structure, pseudogenes, non-protein-coding RNAs, transcribed RNAs, multiple sequence alignment and evolutionarily constrained elements. The ENCODE project at UCSC website (http://genome.ucsc.edu/ENCODE) is the primary portal for the sequence-based data produced as part of the ENCODE project. In the pilot phase of the project, over 30 labs provided experimental results for a total of 56 browser tracks supported by 385 database tables. The site provides researchers with a number of tools that allow them to visualize and analyze the data as well as download data for local analyses. This paper describes the portal to the data, highlights the data that has been made available, and presents the tools that have been developed within the ENCODE project. Access to the data and types of interactive analysis that are possible are illustrated through supplemental examples.

H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

PubMed Central

Hertzberger, Rosanne; Arents, Jos; Dekker, Henk L.; Pridmore, R. David; Gysler, Christof; Kleerebezem, Michiel

2014-01-01

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii. PMID:24487531

The Aspergillus fumigatus septins play pleiotropic roles in septation, conidiation, and cell wall stress, but are dispensable for virulence.

PubMed

Vargas-Muñiz, José M; Renshaw, Hilary; Richards, Amber D; Lamoth, Frédéric; Soderblom, Erik J; Moseley, M Arthur; Juvvadi, Praveen R; Steinbach, William J

2015-08-01

Septins are a conserved family of GTPases that regulate important cellular processes such as cell wall integrity, and septation in fungi. The requirement of septins for virulence has been demonstrated in the human pathogenic yeasts Candida albicans and Cryptococcus neoformans, as well as the plant pathogen Magnaporthe oryzae. Aspergillus spp. contains five genes encoding for septins (aspA-E). While the importance of septins AspA, AspB, AspC, and AspE for growth and conidiation has been elucidated in the filamentous fungal model Aspergillus nidulans, nothing is known on the role of septins in growth and virulence in the human pathogen Aspergillus fumigatus. Here we deleted all five A. fumigatus septins, and generated certain double and triple septin deletion strains. Phenotypic analyses revealed that while all the septins are dispensable in normal growth conditions, AspA, AspB, AspC and AspE are required for regular septation. Furthermore, deletion of only the core septin genes significantly reduced conidiation. Concomitant with the absence of an electron-dense outer conidial wall, the ΔaspB strain was also sensitive to anti-cell wall agents. Infection with the ΔaspB strain in a Galleria mellonella model of invasive aspergillosis showed hypervirulence, but no virulence difference was noted when compared to the wild-type strain in a murine model of invasive aspergillosis. Although the deletion of aspB resulted in increased release of TNF-α from the macrophages, no significant inflammation differences in lung histology was noted between the ΔaspB strain and the wild-type strain. Taken together, these results point to the importance of septins in A. fumigatus growth, but not virulence in a murine model. Copyright © 2015 Elsevier Inc. All rights reserved.

Interactive searching of facial image databases

NASA Astrophysics Data System (ADS)

Nicholls, Robert A.; Shepherd, John W.; Shepherd, Jean

1995-09-01

A set of psychological facial descriptors has been devised to enable computerized searching of criminal photograph albums. The descriptors have been used to encode image databased of up to twelve thousand images. Using a system called FACES, the databases are searched by translating a witness' verbal description into corresponding facial descriptors. Trials of FACES have shown that this coding scheme is more productive and efficient than searching traditional photograph albums. An alternative method of searching the encoded database using a genetic algorithm is currenly being tested. The genetic search method does not require the witness to verbalize a description of the target but merely to indicate a degree of similarity between the target and a limited selection of images from the database. The major drawback of FACES is that is requires a manual encoding of images. Research is being undertaken to automate the process, however, it will require an algorithm which can predict human descriptive values. Alternatives to human derived coding schemes exist using statistical classifications of images. Since databases encoded using statistical classifiers do not have an obvious direct mapping to human derived descriptors, a search method which does not require the entry of human descriptors is required. A genetic search algorithm is being tested for such a purpose.

Anatomical and functional organization of the human substantia nigra and its connections

PubMed Central

Zhang, Yu; Larcher, Kevin Michel-Herve; Misic, Bratislav

2017-01-01

We investigated the anatomical and functional organization of the human substantia nigra (SN) using diffusion and functional MRI data from the Human Connectome Project. We identified a tripartite connectivity-based parcellation of SN with a limbic, cognitive, motor arrangement. The medial SN connects with limbic striatal and cortical regions and encodes value (greater response to monetary wins than losses during fMRI), while the ventral SN connects with associative regions of cortex and striatum and encodes salience (equal response to wins and losses). The lateral SN connects with somatomotor regions of striatum and cortex and also encodes salience. Behavioral measures from delay discounting and flanker tasks supported a role for the value-coding medial SN network in decisional impulsivity, while the salience-coding ventral SN network was associated with motor impulsivity. In sum, there is anatomical and functional heterogeneity of human SN, which underpins value versus salience coding, and impulsive choice versus impulsive action. PMID:28826495

PURA, the gene encoding Pur-alpha, member of an ancient nucleic acid-binding protein family with mammalian neurological functions.

PubMed

Daniel, Dianne C; Johnson, Edward M

2018-02-15

The PURA gene encodes Pur-alpha, a 322 amino acid protein with repeated nucleic acid binding domains that are highly conserved from bacteria through humans. PUR genes with a single copy of this domain have been detected so far in spirochetes and bacteroides. Lower eukaryotes possess one copy of the PUR gene, whereas chordates possess 1 to 4 PUR family members. Human PUR genes encode Pur-alpha (Pura), Pur-beta (Purb) and two forms of Pur-gamma (Purg). Pur-alpha is a protein that binds specific DNA and RNA sequence elements. Human PURA, located at chromosome band 5q31, is under complex control of three promoters. The entire protein coding sequence of PURA is contiguous within a single exon. Several studies have found that overexpression or microinjection of Pura inhibits anchorage-independent growth of oncogenically transformed cells and blocks proliferation at either G1-S or G2-M checkpoints. Effects on the cell cycle may be mediated by interaction of Pura with cellular proteins including Cyclin/Cdk complexes and the Rb tumor suppressor protein. PURA knockout mice die shortly after birth with effects on brain and hematopoietic development. In humans environmentally induced heterozygous deletions of PURA have been implicated in forms of myelodysplastic syndrome and progression to acute myelogenous leukemia. Pura plays a role in AIDS through association with the HIV-1 protein, Tat. In the brain Tat and Pura association in glial cells activates transcription and replication of JC polyomavirus, the agent causing the demyelination disease, progressive multifocal leukoencephalopathy. Tat and Pura also act to stimulate replication of the HIV-1 RNA genome. In neurons Pura accompanies mRNA transcripts to sites of translation in dendrites. Microdeletions in the PURA locus have been implicated in several neurological disorders. De novo PURA mutations have been related to a spectrum of phenotypes indicating a potential PURA syndrome. The nucleic acid, G-rich Pura binding element is amplified as expanded polynucleotide repeats in several brain diseases including fragile X syndrome and a familial form of amyotrophic lateral sclerosis/fronto-temporal dementia. Throughout evolution the Pura protein plays a critical role in survival, based on conservation of its nucleic acid binding properties. These Pura properties have been adapted in higher organisms to the as yet unfathomable development of the human brain. Copyright © 2017 Elsevier B.V. All rights reserved.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal–Hreidarsson syndrome

PubMed Central

Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J.; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H.; Lieberman, Paul M.; Tzfati, Yehuda

2013-01-01

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal–Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres. PMID:23959892

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

PubMed

Deng, Zhong; Glousker, Galina; Molczan, Aliah; Fox, Alan J; Lamm, Noa; Dheekollu, Jayaraju; Weizman, Orr-El; Schertzer, Michael; Wang, Zhuo; Vladimirova, Olga; Schug, Jonathan; Aker, Memet; Londoño-Vallejo, Arturo; Kaestner, Klaus H; Lieberman, Paul M; Tzfati, Yehuda

2013-09-03

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro.

PubMed

Tran, Thao D; Huynh, Steven; Parker, Craig T; Hnasko, Robert; Gorski, Lisa; McGarvey, Jeffery A

2018-06-21

Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes . Copyright © 2018 Tran et al.

Dopamine modulates episodic memory persistence in old age

PubMed Central

Chowdhury, Rumana; Guitart-Masip, Marc; Bunzeck, Nico; Dolan, Raymond J; Düzel, Emrah

2013-01-01

Activation of the hippocampus is required in order to encode memories for new events (or episodes). Observations from animal studies suggest that for these memories to persist beyond 4 to 6 hours, a release of dopamine generated by strong hippocampal activation is needed. This predicts that dopaminergic enhancement should improve human episodic memory persistence also for events encoded with weak hippocampal activation. Here, using pharmacological fMRI in an elderly population where there is a loss of dopamine neurons as part of normal aging, we show this very effect. The dopamine precursor levodopa led to a dose-dependent (inverted U-shape) persistent episodic memory benefit for images of scenes when tested after 6 hours, independent of whether encoding-related hippocampal fMRI activity was weak or strong (U-shaped dose-response relationship). This lasting improvement even for weakly encoded events supports a role for dopamine in human episodic memory consolidation albeit operating within a narrow dose range. PMID:23055489

Orienting in virtual environments: How are surface features and environmental geometry weighted in an orientation task?

PubMed

Kelly, Debbie M; Bischof, Walter F

2008-10-01

We investigated how human adults orient in enclosed virtual environments, when discrete landmark information is not available and participants have to rely on geometric and featural information on the environmental surfaces. In contrast to earlier studies, where, for women, the featural information from discrete landmarks overshadowed the encoding of the geometric information, Experiment 1 showed that when featural information is conjoined with the environmental surfaces, men and women encoded both types of information. Experiment 2 showed that, although both types of information are encoded, performance in locating a goal position is better if it is close to a geometrically or featurally distinct location. Furthermore, although features are relied upon more strongly than geometry, initial experience with an environment influences the relative weighting of featural and geometric cues. Taken together, these results show that human adults use a flexible strategy for encoding spatial information.

Epigenetics, chromatin and genome organization: recent advances from the ENCODE project.

PubMed

Siggens, L; Ekwall, K

2014-09-01

The organization of the genome into functional units, such as enhancers and active or repressed promoters, is associated with distinct patterns of DNA and histone modifications. The Encyclopedia of DNA Elements (ENCODE) project has advanced our understanding of the principles of genome, epigenome and chromatin organization, identifying hundreds of thousands of potential regulatory regions and transcription factor binding sites. Part of the ENCODE consortium, GENCODE, has annotated the human genome with novel transcripts including new noncoding RNAs and pseudogenes, highlighting transcriptional complexity. Many disease variants identified in genome-wide association studies are located within putative enhancer regions defined by the ENCODE project. Understanding the principles of chromatin and epigenome organization will help to identify new disease mechanisms, biomarkers and drug targets, particularly as ongoing epigenome mapping projects generate data for primary human cell types that play important roles in disease. © 2014 The Association for the Publication of the Journal of Internal Medicine.

Metadata Analysis of Phanerochaete chrysosporium Gene Expression Data Identified Common CAZymes Encoding Gene Expression Profiles Involved in Cellulose and Hemicellulose Degradation.

PubMed

Kameshwar, Ayyappa Kumar Sista; Qin, Wensheng

2017-01-01

In literature, extensive studies have been conducted on popular wood degrading white rot fungus, Phanerochaete chrysosporium about its lignin degrading mechanisms compared to the cellulose and hemicellulose degrading abilities. This study delineates cellulose and hemicellulose degrading mechanisms through large scale metadata analysis of P. chrysosporium gene expression data (retrieved from NCBI GEO) to understand the common expression patterns of differentially expressed genes when cultured on different growth substrates. Genes encoding glycoside hydrolase classes commonly expressed during breakdown of cellulose such as GH-5,6,7,9,44,45,48 and hemicellulose are GH-2,8,10,11,26,30,43,47 were found to be highly expressed among varied growth conditions including simple customized and complex natural plant biomass growth mediums. Genes encoding carbohydrate esterase class enzymes CE (1,4,8,9,15,16) polysaccharide lyase class enzymes PL-8 and PL-14, and glycosyl transferases classes GT (1,2,4,8,15,20,35,39,48) were differentially expressed in natural plant biomass growth mediums. Based on these results, P. chrysosporium, on natural plant biomass substrates was found to express lignin and hemicellulose degrading enzymes more than cellulolytic enzymes except GH-61 (LPMO) class enzymes, in early stages. It was observed that the fate of P. chrysosporium transcriptome is significantly affected by the wood substrate provided. We believe, the gene expression findings in this study plays crucial role in developing genetically efficient microbe with effective cellulose and hemicellulose degradation abilities.

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

PubMed Central

Eraso, Jesus M.; Olsen, Randall J.; Beres, Stephen B.; Kachroo, Priyanka; Porter, Adeline R.; Nasser, Waleed; Bernard, Paul E.; DeLeo, Frank R.

2016-01-01

To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here. PMID:27600505

The EWS–Oct-4 fusion gene encodes a transforming gene

PubMed Central

Lee, Jungwoon; Kim, Ja Young; Kang, In Young; Kim, Hye Kyoung; Han, Yong-Mahn; Kim, Jungho

2007-01-01

The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS–Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS–Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS–Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS–Oct-4 are necessary for full transactivation potential. EWS–Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS–Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS–Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product. PMID:17564582

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse

PubMed Central

2011-01-01

Background Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB). Results Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB. Conclusions Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. PMID:22008461

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

PubMed

Kamenskaya, D N; Pankova, M V; Atopkin, D M; Brykov, V A

2017-01-01

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

EsrE-A yigP Locus-Encoded Transcript-Is a 3′ UTR sRNA Involved in the Respiratory Chain of E. coli

PubMed Central

Xia, Hui; Yang, Xichen; Tang, Qiongwei; Ye, Jiang; Wu, Haizhen; Zhang, Huizhan

2017-01-01

The yigP locus is widely conserved among γ-proteobacteria. Mutation of the yigP locus impacts aerobic growth of Gram-negative bacteria. However, the underlying mechanism of how the yigP locus influences aerobic growth remains largely unknown. Here, we demonstrated that the yigP locus in Escherichia coli encodes two transcripts; the mRNA of ubiquinone biosynthesis protein, UbiJ, and the 3′ untranslated region small regulatory RNA (sRNA), EsrE. EsrE is an independent transcript that is transcribed using an internal promoter of the yigP locus. Surprisingly, we found that both the EsrE sRNA and UbiJ protein were required for Q8 biosynthesis, and were sufficient to rescue the growth defect ascribed to deletion of the yigP locus. Moreover, our data showed that EsrE targeted multiple mRNAs involved in several cellular processes including murein biosynthesis and the tricarboxylic acid cycle. Among these targets, sdhD mRNA that encodes one subunit of succinate dehydrogenase (SDH), was significantly activated. Our findings provided an insight into the important function of EsrE in bacterial adaptation to various environments, as well as coordinating different aspects of bacterial physiology. PMID:28900423

Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae

PubMed Central

Berghofer, Y.; Klein, A.

1995-01-01

Methanococcus voltae, which contains four different gene groups that encode [NiFe]-hydrogenases, was transformed with integration vectors to achieve polar inactivation of two of the four hydrogenase operons that encode the selenium-free enzymes Vhc and Frc. Transformants which were selected by their acquired puromycin resistance showed site-specific insertions in either the vhc or frc operon by single crossover events. Southern hybridization revealed tandem integrations of whole vectors in the vhc operon, whereas only one vector copy was found in the frc operon. Northern (RNA) hybridizations showed a pac transcript of defined size, indicating strong termination in front of the hydrogenase genes downstream. In spite of the apparent abolition of expression of selenium-free hydrogenases through these polar insertions, they were not lethal to cells upon growth in selenium-deprived minimal medium, which we had previously shown to strongly induce transcription of the respective operons in M. voltae. Instead, like wild-type control cultures, transformants responded to selenium deprivation only with a reduction in growth rate. We conclude that loss of the potential to express a selenium-free hydrogenase can nevertheless be balanced by very small amounts of selenium hydrogenases under laboratory conditions in which the hydrogen supply is not likely to be a limiting growth factor. PMID:16535019

Cloning, expression and characterization of a novel human CAP10-like gene hCLP46 from CD34(+) stem/progenitor cells.

PubMed

Teng, Yun; Liu, Qiaohong; Ma, Jie; Liu, Feng; Han, Zeguang; Wang, Youxin; Wang, Wei

2006-04-12

A novel human gene, named as human CAP10-like protein 46 kDa (hCLP46), was isolated and identified from human acute myeloid leukemia transformed from myelodysplastic syndrome (MDS-AML) CD34(+) cells. hCLP46 (3q13.33) contains 11 exons encoding a putative protein of 392 amino acids, with a highly conserved CAP10 domain, a hydrophobic signal peptide at its N-terminus, and an endoplasmic reticulum (ER) retention signal motif KTEL at the C-terminus. The homologs of hCLP46 exist in different organisms from plants to animal kingdoms. Subcellular localization analysis showed that hCLP46 is an ER-resident protein. hCLP46 expressed in most human adult tissues at different intensities, with lengths of 3.5 kb and 1.9 kb. Transcript of hCLP46 was not detectable in colon, thymus, and small intestine, but was abundant in liver, indicating that hCLP46 may be involved in important physiological functions in the liver. hCLP46 over-expressed U937 cells had higher growth rate than the cells without exogenic hCLP46 protein expression, suggesting that hCLP46 protein possess the ability of promoting cell proliferation.

Plasmonic Encoding

DTIC Science & Technology

2014-10-06

The nanosheets, like many SERS platforms, are ideally suited for encoding schemes based on the SERS signal from a variety of thiolated small...counterfeiting purposes. The nanosheets, like many SERS platforms, are ideally suited for encoding schemes based on the SERS signal from a variety of...environments ( like the surface of human hair). 2. Nanoflares In 2007, we first introduced the concept of nanoflares. Nanoflares are a new class of

Effects of Δ9-tetrahydrocannabinol administration on human encoding and recall memory function: a pharmacological FMRI study.

PubMed

Bossong, Matthijs G; Jager, Gerry; van Hell, Hendrika H; Zuurman, Lineke; Jansma, J Martijn; Mehta, Mitul A; van Gerven, Joop M A; Kahn, René S; Ramsey, Nick F

2012-03-01

Deficits in memory function are an incapacitating aspect of various psychiatric and neurological disorders. Animal studies have recently provided strong evidence for involvement of the endocannabinoid (eCB) system in memory function. Neuropsychological studies in humans have shown less convincing evidence but suggest that administration of cannabinoid substances affects encoding rather than recall of information. In this study, we examined the effects of perturbation of the eCB system on memory function during both encoding and recall. We performed a pharmacological MRI study with a placebo-controlled, crossover design, investigating the effects of Δ9-tetrahydrocannabinol (THC) inhalation on associative memory-related brain function in 13 healthy volunteers. Performance and brain activation during associative memory were assessed using a pictorial memory task, consisting of separate encoding and recall conditions. Administration of THC caused reductions in activity during encoding in the right insula, the right inferior frontal gyrus, and the left middle occipital gyrus and a network-wide increase in activity during recall, which was most prominent in bilateral cuneus and precuneus. THC administration did not affect task performance, but while during placebo recall activity significantly explained variance in performance, this effect disappeared after THC. These findings suggest eCB involvement in encoding of pictorial information. Increased precuneus activity could reflect impaired recall function, but the absence of THC effects on task performance suggests a compensatory mechanism. These results further emphasize the eCB system as a potential novel target for treatment of memory disorders and a promising target for development of new therapies to reduce memory deficits in humans.

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

PubMed Central

Nishibuchi, M; Murakami, A; Arita, M; Jikuya, H; Takano, J; Honda, T; Miwatani, T

1989-01-01

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay. PMID:2685027

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

PubMed Central

Weekes, M. P.; Antrobus, R.; Rorbach, J.; van Haute, L.; Umrania, Y.; Smith, D. L.; Minczuk, M.; Lehner, P. J.; Sinclair, J. H.

2016-01-01

ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. PMID:27025248

The Role of CHD7 Mutations in Patients with Idiopathic Hypogonadotropic Hypogonadism and Kallmann Syndrome

PubMed Central

Kim, Hyung-Goo; Layman, Lawrence C.

2013-01-01

Mutations in the chromodomain helicase DNA binding protein-7 (CHD7) cause CHARGE syndrome, which includes eye coloboma, heart malformations, atresia of the choanae, retardation of growth/development, genital anomalies, and ear abnormalities. CHARGE syndrome is usually sporadic, but is also autosomal dominant. CHD7 encodes a large protein that participates in chromatin remodeling and transcription. Findings from studies of mouse models employing ENU-mutagenesis or gene-trap methods recapitulate human CHARGE syndrome. CHARGE patients may manifest anosmia and/or hypogonadism, features that overlap with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). Similarly, IHH/KS patients may also display partial CHARGE features. Therefore, it has been hypothesized that IHH/KS represents a milder allelic variant of CHARGE syndrome, which has been supported by the identification of heterozygous CHD7 mutations in both normosmic IHH and KS. Developmental expression within the hypothalamus and the presence of human mutations indicate that CHD7 has an important role in puberty and reproduction. PMID:21856375

Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

PubMed

Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

2016-10-01

Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

RNA sequencing analysis of human podocytes reveals glucocorticoid regulated gene networks targeting non-immune pathways

PubMed Central

Jiang, Lulu; Hindmarch, Charles C. T.; Rogers, Mark; Campbell, Colin; Waterfall, Christy; Coghill, Jane; Mathieson, Peter W.; Welsh, Gavin I.

2016-01-01

Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or ‘podocytes’, the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes. PMID:27774996

A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

PubMed Central

Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

2002-01-01

A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

Tracking Temporal Hazard in the Human Electroencephalogram Using a Forward Encoding Model

PubMed Central

2018-01-01

Abstract Human observers automatically extract temporal contingencies from the environment and predict the onset of future events. Temporal predictions are modeled by the hazard function, which describes the instantaneous probability for an event to occur given it has not occurred yet. Here, we tackle the question of whether and how the human brain tracks continuous temporal hazard on a moment-to-moment basis, and how flexibly it adjusts to strictly implicit variations in the hazard function. We applied an encoding-model approach to human electroencephalographic data recorded during a pitch-discrimination task, in which we implicitly manipulated temporal predictability of the target tones by varying the interval between cue and target tone (i.e. the foreperiod). Critically, temporal predictability either was driven solely by the passage of time (resulting in a monotonic hazard function) or was modulated to increase at intermediate foreperiods (resulting in a modulated hazard function with a peak at the intermediate foreperiod). Forward-encoding models trained to predict the recorded EEG signal from different temporal hazard functions were able to distinguish between experimental conditions, showing that implicit variations of temporal hazard bear tractable signatures in the human electroencephalogram. Notably, this tracking signal was reconstructed best from the supplementary motor area, underlining this area’s link to cognitive processing of time. Our results underline the relevance of temporal hazard to cognitive processing and show that the predictive accuracy of the encoding-model approach can be utilized to track abstract time-resolved stimuli. PMID:29740594

Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas.

PubMed

Calin, George A; Liu, Chang-gong; Ferracin, Manuela; Hyslop, Terry; Spizzo, Riccardo; Sevignani, Cinzia; Fabbri, Muller; Cimmino, Amelia; Lee, Eun Joo; Wojcik, Sylwia E; Shimizu, Masayoshi; Tili, Esmerina; Rossi, Simona; Taccioli, Cristian; Pichiorri, Flavia; Liu, Xiuping; Zupo, Simona; Herlea, Vlad; Gramantieri, Laura; Lanza, Giovanni; Alder, Hansjuerg; Rassenti, Laura; Volinia, Stefano; Schmittgen, Thomas D; Kipps, Thomas J; Negrini, Massimo; Croce, Carlo M

2007-09-01

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

IGF-I stimulates ERβ and aromatase expression via IGF1R/PI3K/AKT-mediated transcriptional activation in endometriosis.

PubMed

Zhou, Yan; Zeng, Cheng; Li, Xin; Wu, Pei-Li; Yin, Ling; Yu, Xiao-Lan; Zhou, Ying-Fang; Xue, Qing

2016-08-01

Estrogen receptor beta (ERβ, encoded by ESR2 gene) and cytochrome P450 aromatase (encoded by CYP19A1 gene) play critical roles in endometriosis, and the levels of insulin-like growth factor-I (IGF-I) in the peritoneal fluid are significantly higher in patients with endometriosis compared with those in normal women. However, the effects and mechanisms of IGF-I on ERβ and aromatase expression remain to be fully elucidated. In this study, human endometriotic stromal cells (ESCs) and endometrial cells (EMs) derived from ovarian endometriomas and eutopic endometrial tissues. ESCs were cultured with IGF-I, signal pathway inhibitors, and siRNAs. ERβ and aromatase expression were measured by real-time PCR and Western, respectively. The binding of c-Jun and CREB to the ESR2 and CYP19A1 promoters was assessed by chromatin immunoprecipitation assay. Animal experiments were performed in a xenograft mouse model. Levels of IGF-I mRNA in ESCs were markedly higher than those in EMs. IGF-I upregulated ERβ and aromatase expression in ESCs after stimulation of the IGF1R/PI3K/AKT pathway. Following IGF-I treatment, a marked increase in c-Jun and CREB phosphorylation occurred, enhancing binding to the ESR2 and CYP19A1 promoters. An IGF1R inhibitor in vivo reduced IGF-I-induced endometriosis graft growth and ERβ and aromatase expression. In conclusion, this is the first report to describe a mechanistic analysis of ERβ and aromatase expression regulated by IGF-I in ESCs. Moreover, an IGF1R inhibitor impeded ectopic lesion growth in nude mice. These findings suggest that an inhibitor of IGF1R might have therapeutic potential as an antiendometriotic drug. Level of IGF-I mRNA in ESCs is markedly higher than that in EMs. IGF-I up-regulates ERβ and aromatase expression via IGF1R/PI3K/AKT pathway. C-Jun and CREB are recruited to ESR2 or CYP19A1 promoter by IGF-I stimulation. IGF-1R inhibitors in vivo impede the growth of ectopic lesions in nude mice.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5).

PubMed

Müller, Miriam; Persson, Anja Bondke; Krueger, Katharina; Kirschner, Karin M; Scholz, Holger

2017-07-01

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(-KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(-KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(-KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(-KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology. Copyright © 2017 Elsevier B.V. All rights reserved.

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism, acid tolerance response and biofilm formation.

PubMed

Liao, S; Bitoun, J P; Nguyen, A H; Bozner, D; Yao, X; Wen, Z T

2015-08-01

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA-binding N-terminal domain and a C-terminal domain highly homologous to the pyridoxal phosphate-dependent aspartate aminotransferases. A pdxR-deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR-deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real-time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR-deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6 . Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Activation of the FGFR1 signalling pathway by the Epstein-Barr virus-encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells.

PubMed

Lo, Angela Kwok-Fung; Dawson, Christopher W; Young, Lawrence S; Ko, Chuen-Wai; Hau, Pok-Man; Lo, Kwok-Wai

2015-10-01

Non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c-Myc, and HIF-1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up-regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1-mediated aerobic glycolysis, cellular transformation (proliferation and anchorage-independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1-mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage-independent growth of NPC cells. Our current findings demonstrate that LMP1-mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV-driven pathogenesis of NPC. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effects of H2 and Formate on Growth Yield and Regulation of Methanogenesis in Methanococcus maripaludis

PubMed Central

Costa, Kyle C.; Yoon, Sung Ho; Pan, Min; Burn, June A.; Baliga, Nitin S.

2013-01-01

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. PMID:23335420

Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression.

PubMed Central

Jonas, V; Lin, C R; Kawashima, E; Semon, D; Swanson, L W; Mermod, J J; Evans, R M; Rosenfeld, M G

1985-01-01

Two mRNAs generated as a consequence of alternative RNA processing events in expression of the human calcitonin gene encode the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Both calcitonin and CGRP RNAs and their encoded peptide products are expressed in the human pituitary and in medullary thyroid tumors. On the basis of sequence comparison, it is suggested that both the calcitonin and CGRP exons arose from a common primordial sequence, suggesting that duplication and rearrangement events are responsible for the generation of this complex transcription unit. Images PMID:3872459

Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

PubMed

Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

2000-05-31

cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters.

PubMed

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

PubMed Central

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

The importance of neuronal growth factors in the ovary.

PubMed

Streiter, S; Fisch, B; Sabbah, B; Ao, A; Abir, R

2016-01-01

The neurotrophin family consists of nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4/5 (NT4/5), in addition to brain-derived neurotrophic factor (BDNF) and the neuronal growth factors, glial cell line-derived neurotrophic factor (GDNF) and vasointestinal peptide (VIP). Although there are a few literature reviews, mainly of animal studies, on the importance of neurotrophins in the ovary, we aimed to provide a complete review of neurotrophins as well as neuronal growth factors and their important roles in normal and pathological processes in the ovary. Follicular assembly is probably stimulated by complementary effects of NGF, NT4/5 and BDNF and their receptors. The neurotrophins, GDNF and VIP and their receptors have all been identified in preantral and antral follicles of mammalian species, including humans. Transgenic mice with mutations in the genes encoding for Ngf, Nt4/5 and Bdnf and their tropomyosin-related kinase β receptor showed a reduction in preantral follicles and an abnormal ovarian morphology, whereas NGF, NT3, GDNF and VIP increased the in vitro activation of primordial follicles in rats and goats. Additionally, NGF, NT3 and GDNF promoted follicular cell proliferation; NGF, BDNF and VIP were shown to be involved in ovulation; VIP inhibited follicular apoptosis; NT4/5, BDNF and GDNF promoted oocyte maturation and NGF, NT3 and VIP stimulated steroidogenesis. NGF may also exert a stimulatory effect in ovarian cancer and polycystic ovarian syndrome (PCOS). Low levels of NGF and BDNF in follicular fluid may be associated with diminished ovarian reserve and high levels with endometriosis. More knowledge of the roles of neuronal growth factors in the ovary has important implications for the development of new therapeutic drugs (such as anti-NGF agents) for ovarian cancer and PCOS as well as various infertility problems, warranting further research. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Macrocyclic lactones differ in interaction with recombinant P-glycoprotein 9 of the parasitic nematode Cylicocylus elongatus and ketoconazole in a yeast growth assay.

PubMed

Kaschny, Maximiliane; Demeler, Janina; Janssen, I Jana I; Kuzmina, Tetiana A; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2015-04-01

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner.

Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic Nematode Cylicocylus elongatus and Ketoconazole in a Yeast Growth Assay

PubMed Central

Kaschny, Maximiliane; Demeler, Janina; Janssen, I. Jana I.; Kuzmina, Tetiana A.; Besognet, Bruno; Kanellos, Theo; Kerboeuf, Dominique; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2015-01-01

Macrocyclic lactones (MLs) are widely used parasiticides against nematodes and arthropods, but resistance is frequently observed in parasitic nematodes of horses and livestock. Reports claiming resistance or decreased susceptibility in human nematodes are increasing. Since no target site directed ML resistance mechanisms have been identified, non-specific mechanisms were frequently implicated in ML resistance, including P-glycoproteins (Pgps, designated ABCB1 in vertebrates). Nematode genomes encode many different Pgps (e.g. 10 in the sheep parasite Haemonchus contortus). ML transport was shown for mammalian Pgps, Pgps on nematode egg shells, and very recently for Pgp-2 of H. contortus. Here, Pgp-9 from the equine parasite Cylicocyclus elongatus (Cyathostominae) was expressed in a Saccharomyces cerevisiae strain lacking seven endogenous efflux transporters. Pgp was detected on these yeasts by flow cytometry and chemiluminescence using the monoclonal antibody UIC2, which is specific for the active Pgp conformation. In a growth assay, Pgp-9 increased resistance to the fungicides ketoconazole, actinomycin D, valinomycin and daunorubicin, but not to the anthelmintic fungicide thiabendazole. Since no fungicidal activity has been described for MLs, their interaction with Pgp-9 was investigated in an assay involving two drugs: Yeasts were incubated with the highest ketoconazole concentration not affecting growth plus increasing concentrations of MLs to determine competition between or modulation of transport of both drugs. Already equimolar concentrations of ivermectin and eprinomectin inhibited growth, and at fourfold higher ML concentrations growth was virtually abolished. Selamectin and doramectin did not increase susceptibility to ketoconazole at all, although doramectin has been shown previously to strongly interact with human and canine Pgp. An intermediate interaction was observed for moxidectin. This was substantiated by increased binding of UIC2 antibodies in the presence of ivermectin, moxidectin, daunorubicin and ketoconazole but not selamectin. These results demonstrate direct effects of MLs on a recombinant nematode Pgp in an ML-specific manner. PMID:25849454

Enhanced Tumor Growth and Invasiveness in Vivo by a Carboxyl-Terminal Fragment of α1-Proteinase Inhibitor Generated by Matrix Metalloproteinases

PubMed Central

Kataoka, Hiroaki; Uchino, Hirofumi; Iwamura, Takeshi; Seiki, Motoharu; Nabeshima, Kazuki; Koono, Masashi

1999-01-01

Matrix metalloproteinases (MMPs) are believed to contribute to the complex process of cancer progression. They also exhibit an α1-proteinase inhibitor (αPI)-degrading activity generating a carboxyl-terminal fragment of ∼5 kd (αPI-C). This study reports that overexpression of αPI-C in S2–020, a cloned subline derived from the human pancreas adenocarcinoma cell line SUIT-2, potentiates the growth capability of the cells in nude mice. After stable transfection of a vector containing a chimeric cDNA encoding a signal peptide sequence of tissue inhibitor of metalloproteinase-1 followed by cDNA for αPI-C into S2–020 cells, three clones that stably secrete αPI-C were obtained. The ectopic expression of αPI-C did not alter in vitro cellular growth. However, subcutaneous injection of the αPI-C-secreting clones resulted in tumors that were 1.5 to 3-fold larger than those of control clones with an increased tendency to invasiveness and lymph node metastasis. These effects could be a result of modulation of natural killer (NK) cell-mediated control of tumor growth in nude mice, as the growth advantage of αPI-C-secreting clones was not observed in NK-depleted mice, and αPI-C-secreting clones showed decreased NK sensitivity in vitro. In addition, production of αPI and generation of the cleaved form of αPI by MMP were observed in various human tumor cell lines and in a highly metastatic subline of SUIT-2 in vitro. These results provide experimental evidence that the αPI-degrading activity of MMPs may play a role in tumor progression not only via the inactivation of αPI but also via the generation of αPI-C. PMID:10027404

Differential Expression of CHL1 Gene during Development of Major Human Cancers

PubMed Central

Senchenko, Vera N.; Krasnov, George S.; Dmitriev, Alexey A.; Kudryavtseva, Anna V.; Anedchenko, Ekaterina A.; Braga, Eleonora A.; Pronina, Irina V.; Kondratieva, Tatiana T.; Ivanov, Sergey V.; Zabarovsky, Eugene R.; Lerman, Michael I.

2011-01-01

Background CHL1 gene (also known as CALL) on 3p26.3 encodes a one-pass trans-membrane cell adhesion molecule (CAM). Previously CAMs of this type, including L1, were shown to be involved in cancer growth and metastasis. Methodology/Principal Findings We used Clontech Cancer Profiling Arrays (19 different types of cancers, 395 samples) to analyze expression of the CHL1 gene. The results were further validated by RT-qPCR for breast, renal and lung cancer. Cancer Profiling Arrays revealed differential expression of the gene: down-regulation/silencing in a majority of primary tumors and up-regulation associated with invasive/metastatic growth. Frequent down-regulation (>40% of cases) was detected in 11 types of cancer (breast, kidney, rectum, colon, thyroid, stomach, skin, small intestine, bladder, vulva and pancreatic cancer) and frequent up-regulation (>40% of cases) – in 5 types (lung, ovary, uterus, liver and trachea) of cancer. Using real-time quantitative PCR (RT-qPCR) we found that CHL1 expression was decreased in 61% of breast, 60% of lung, 87% of clear cell and 89% papillary renal cancer specimens (P<0.03 for all the cases). There was a higher frequency of CHL1 mRNA decrease in lung squamous cell carcinoma compared to adenocarcinoma (81% vs. 38%, P = 0.02) without association with tumor progression. Conclusions/Significance Our results suggested that CHL1 is involved in the development of different human cancers. Initially, during the primary tumor growth CHL1 could act as a putative tumor suppressor and is silenced to facilitate in situ tumor growth for 11 cancer types. We also suggested that re-expression of the gene on the edge of tumor mass might promote local invasive growth and enable further metastatic spread in ovary, colon and breast cancer. Our data also supported the role of CHL1 as a potentially novel specific biomarker in the early pathogenesis of two major histological types of renal cancer. PMID:21408220

Brain computer interface to enhance episodic memory in human participants

PubMed Central

Burke, John F.; Merkow, Maxwell B.; Jacobs, Joshua; Kahana, Michael J.

2015-01-01

Recent research has revealed that neural oscillations in the theta (4–8 Hz) and alpha (9–14 Hz) bands are predictive of future success in memory encoding. Because these signals occur before the presentation of an upcoming stimulus, they are considered stimulus-independent in that they correlate with enhanced memory encoding independent of the item being encoded. Thus, such stimulus-independent activity has important implications for the neural mechanisms underlying episodic memory as well as the development of cognitive neural prosthetics. Here, we developed a brain computer interface (BCI) to test the ability of such pre-stimulus activity to modulate subsequent memory encoding. We recorded intracranial electroencephalography (iEEG) in neurosurgical patients as they performed a free recall memory task, and detected iEEG theta and alpha oscillations that correlated with optimal memory encoding. We then used these detected oscillatory changes to trigger the presentation of items in the free recall task. We found that item presentation contingent upon the presence of pre-stimulus theta and alpha oscillations modulated memory performance in more sessions than expected by chance. Our results suggest that an electrophysiological signal may be causally linked to a specific behavioral condition, and contingent stimulus presentation has the potential to modulate human memory encoding. PMID:25653605

Multiscale Models in the Biomechanics of Plant Growth

PubMed Central

Fozard, John A.

2015-01-01

Plant growth occurs through the coordinated expansion of tightly adherent cells, driven by regulated softening of cell walls. It is an intrinsically multiscale process, with the integrated properties of multiple cell walls shaping the whole tissue. Multiscale models encode physical relationships to bring new understanding to plant physiology and development. PMID:25729061

Phylogeny of fungal hemoglobins and expression analysis of the Aspergillus oryzae flavohemoglobin gene fhbA during hyphal growth.

PubMed

te Biesebeke, Rob; Levasseur, Anthony; Boussier, Amandine; Record, Eric; van den Hondel, Cees A M J J; Punt, Peter J

2010-01-01

The fhbA genes encoding putative flavohemoglobins (FHb) from Aspergillus niger and Aspergillus oryzae were isolated. Comparison of the deduced amino acid sequence of the A. niger fhbA gene and other putative filamentous fungal FHb-encoding genes to that of Ralstonia eutropha shows an overall conserved gene structure and completely conserved catalytic amino acids. Several yeasts and filamentous fungi, including both Aspergillus species have been found to contain a small FHb gene family mostly consisting of two family members. Based on these sequences the evolutionary history of the fungal FHb family was reconstructed. The isolated fhbA genes from A. oryzae and A. niger belong to a phylogenetic group, which exclusively contains Aspergillus genes. Different experimental approaches show that fhbA transcript levels appear during active hyphal growth. Moreover, in a pclA-disrupted strain with a hyperbranching growth phenotype, the transcript levels of the fhbA gene were 2–5 times higher compared to the wild-type. These results suggest that FHb from filamentous fungi have a function that is correlated to the hyphal growth phenotype.

Inhibitory effect of streptococci on the growth of M. catarrhalis strains and the diversity of putative bacteriocin-like gene loci in the genomes of S. pneumoniae and its relatives.

PubMed

Ikryannikova, L N; Malakhova, M V; Lominadze, G G; Karpova, I Yu; Kostryukova, E S; Mayansky, N A; Kruglov, A N; Klimova, E A; Lisitsina, E S; Ilina, E N; Govorun, V M

2017-12-13

S. pneumoniae is a facultative human pathogen causing a wide range of infections including the life-threatening pneumoniae or meningitis. It colonizes nasopharynx as well as its closest phylogenetic relatives S. pseudopneumoniae and S. mitis. Both the latter, despite the considerable morphological and phenotypic similarity with the pneumococcus, are considerably less pathogenic for humans and cause infections mainly in the immunocompromized hosts. In this work, we compared the inhibitory effect of S. pneumoniae and its relatives on the growth of Moraxella catarrhalis strains using the culture-based antagonistic test. We observed that the inhibitory effect of S. mitis strains is kept when a hydrogen peroxide produced by cells is inactivated by catalase, and even when the live cells are killed in chloroform vapors, in contrast to the pneumococcus whose inhibiting ability disappeared when the cells die. It was suggested that this effect may be due to the production of bacterial antimicrobial peptides by S. mitis, so we examined the genomes of our strains for the presence of bacteriocin-like peptides encoding genes. We observed that a set of bacteriocin-like genes in the genome of S. mitis is greatly poorer in comparison with S. pneumoniae one; moreover, in one S. mitis strain we found no bacteriocin-like genes. It could mean that there are probably some additional opportunities of S. mitis to inhibit the growth of competing neighbors which are still have to be discovered.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

PubMed Central

Ravi, R K; Weber, E; McMahon, M; Williams, J R; Baylin, S; Mal, A; Harter, M L; Dillehay, L E; Claudio, P P; Giordano, A; Nelkin, B D; Mabry, M

1998-01-01

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors. PMID:9421477

Primary human osteoblasts grow into porous tantalum and maintain an osteoblastic phenotype.

PubMed

Welldon, Katie J; Atkins, Gerald J; Howie, Donald W; Findlay, David M

2008-03-01

Porous tantalum (Ta) has found application in orthopedics, although the interaction of human osteoblasts (HOB) with this material has not been reported. The aim of this study was to investigate the interaction of primary HOB with porous tantalum, using 5-mm thick discs of porous tantalum. Comparison was made with discs of solid tantalum and tissue culture plastic. Confocal microscopy was used to investigate the attachment and growth of cells on porous Ta, and showed that HOB attached successfully to the metal "trabeculae," underwent extensive cell division, and penetrated into the Ta pores. The maturation of HOB on porous Ta was determined in terms of cell expression of the osteoblast phenotypic markers, STRO-1, and alkaline phosphatase. Despite some donor-dependent variation in STRO-1/AlkPhos expression, growth of cells grown on porous Ta either promoted, or did not impede, the maturation of HOB. In addition, the expression of key osteoblastic genes was investigated after 14 days of culture. The relative levels of mRNA encoding osteocalcin, osteopontin and receptor activator of NFkappaB ligand (RANKL) was not different between porous or solid Ta or plastic, although these genes were expressed differently by cells of different donors. However, bone sialoprotein and type I collagen mRNA species showed a decreased expression on porous Ta compared with expression on plastic. No substrate-dependent differences were seen in the extent of in vitro mineralization by HOB. These results indicate that porous Ta is a good substrate for the attachment, growth, and differentiated function of HOB. (c) 2007 Wiley Periodicals, Inc.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates

PubMed Central

Garrido, Daniel; Ruiz-Moyano, Santiago; Jimenez-Espinoza, Rogelio; Eom, Hyun-Ju; Block, David E.; Mills, David A.

2013-01-01

Prebiotics are non-digestible substrates that stimulate the growth of beneficial microbial populations in the intestine, especially Bifidobacterium species. Among them, fructo- and galacto-oligosaccharides are commonly used in the food industry, especially as a supplement for infant formulas. Mechanistic details on the enrichment of bifidobacteria by these prebiotics are important to understand the effects of these dietary interventions. In this study the consumption of galactooligosaccharides was studied for 22 isolates of Bifidobacterium longum subsp. infantis, one of the most representative species in the infant gut microbiota. In general all isolates showed a vigorous growth on these oligosaccharides, but consumption of larger galactooligosaccharides was variable. Bifidobacterium infantis ATCC 15697 has five genes encoding β-galactosidases, and three of them were induced during bacterial growth on commercial galactooligosaccharides. Recombinant β-galactosidases from B. infantis ATCC 15697 displayed different preferences for β-galactosides such as 4′ and 6′-galactobiose, and four β-galactosidases in this strain released monosaccharides from galactooligosaccharides. Finally, we determined the amounts of short chain fatty acids produced by strain ATCC 15697 after growth on different prebiotics. We observed that biomass and product yields of substrate were higher for lactose and galactooligosaccharides, but the amount of acids produced per cell was larger after growth on human milk oligosaccharides. These results provide a molecular basis for galactooligosaccharide consumption in B. infantis, and also represent evidence for physiological differences in the metabolism of prebiotics that might have a differential impact on the host. PMID:23200660

Major contribution of the type II beta carbonic anhydrase CanB (Cj0237) to the capnophilic growth phenotype of Campylobacter jejuni.

PubMed

Al-Haideri, Halah; White, Michael A; Kelly, David J

2016-02-01

Campylobacter jejuni, the leading cause of human bacterial gastroenteritis, requires low environmental oxygen and high carbon dioxide for optimum growth, but the molecular basis for the carbon dioxide requirement is unclear. One factor may be inefficient conversion of gaseous CO2 to bicarbonate, the required substrate of various carboxylases. Two putative carbonic anhydrases (CAs) are encoded in the genome of C. jejuni strain NCTC 11168 (Cj0229 and Cj0237). Here, we show that the deletion of the cj0237 (canB) gene alone prevents growth in complex media at low (1% v/v) CO2 and significantly reduces the growth rate at high (5% v/v) CO2. In minimal media incubated under high CO2, the canB mutant grew on L-aspartate but not on the key C3 compounds L-serine, pyruvate and L-lactate, showing that CanB is crucial in bicarbonate provision for pyruvate carboxylase-mediated oxaloacetate synthesis. Nevertheless, purified CanB (a dimeric, anion and acetazolamide sensitive, zinc-containing type II beta-class enzyme) hydrates CO2 actively only above pH 8 and with a high Km (∼ 34 mM). At typical cytoplasmic pH values and low CO2, these kinetic properties might limit intracellular bicarbonate availability. Taken together, our data suggest CanB is a major contributor to the capnophilic growth phenotype of C. jejuni. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

RASSF10 is epigenetically silenced and functions as a tumor suppressor in gastric cancer

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

Wei, Ziran; Chen, Xia; Chen, Ji

2013-03-22

Highlights: ► Epigenetic silencing of RASSF10 gene expression in GC cells. ► RASSF10 overexpression inhibits cell growth in vitro and in vivo. ► RASSF10 induces apoptosis in GC cells. ► RASSF10 inhibits Wnt/β-catenin signaling pathway. -- Abstract: Ras association domain family (RASSF) proteins are encoded by several tumor suppressor genes that are frequently silenced in human cancers. In this study, we investigated RASSF10 as a target of epigenetic inactivation and examined its functions as a tumor suppressor in gastric cancer. RASSF10 was silenced in six out of eight gastric cancer cell lines. Loss or downregulation of RASSF10 expression was associatedmore » with promoter hypermethylation, and could be restored by a demethylating agent. Overexpression of RASSF10 in gastric cancer cell lines (JRST, BGC823) suppressed cell growth and colony formation, and induced apoptosis, whereas RASSF10 depletion promoted cell growth. In xenograft animal experiments, RASSF10 overexpression effectively repressed tumor growth. Mechanistic investigations revealed that RASSF10 inhibited tumor growth by blocking activation of β-catenin and its downstream targets including c-Myc, cyclinD1, cyclinE1, peroxisome proliferator-activated receptor δ, transcription factor 4, transcription factor 1 and CD44. In conclusion, the results of this study provide insight into the role of RASSF10 as a novel functional tumor suppressor in gastric cancer through inhibition of the Wnt/β-catenin signaling pathway.« less

Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

PubMed

Nieves-Morión, Mercedes; Flores, Enrique

2018-02-01

Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

PubMed

Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

2015-09-01

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

Slow-Theta-to-Gamma Phase–Amplitude Coupling in Human Hippocampus Supports the Formation of New Episodic Memories

PubMed Central

Lega, Bradley; Burke, John; Jacobs, Joshua; Kahana, Michael J.

2016-01-01

Phase–amplitude coupling (PAC) has been proposed as a neural mechanism for coordinating information processing across brain regions. Here we sought to characterize PAC in the human hippocampus, and in temporal and frontal cortices, during the formation of new episodic memories. Intracranial recordings taken as 56 neurosurgical patients studied and recalled lists of words revealed significant hippocampal PAC, with slow-theta activity (2.5–5 Hz) modulating gamma band activity (34–130 Hz). Furthermore, a significant number of hippocampal electrodes exhibited greater PAC during successful than unsuccessful encoding, with the gamma activity at these sites coupled to the trough of the slow-theta oscillation. These same conditions facilitate LTP in animal models, providing a possible mechanism of action for this effect in human memory. Uniquely in the hippocampus, phase preference during item encoding exhibited a biphasic pattern. Overall, our findings help translate between the patterns identified during basic memory tasks in animals and those present during complex human memory encoding. We discuss the unique properties of human hippocampal PAC and how our findings relate to influential theories of information processing based on theta–gamma interactions. PMID:25316340

Prolonged Growth Hormone/Insulin/Insulin-like Growth Factor Nutrient Response Signaling Pathway as a Silent Killer of Stem Cells and a Culprit in Aging.

PubMed

Ratajczak, Mariusz Z; Bartke, Andrzej; Darzynkiewicz, Zbigniew

2017-08-01

The dream of slowing down the aging process has always inspired mankind. Since stem cells are responsible for tissue and organ rejuvenation, it is logical that we should search for encoded mechanisms affecting life span in these cells. However, in adult life the hierarchy within the stem cell compartment is still not very well defined, and evidence has accumulated that adult tissues contain rare stem cells that possess a broad trans-germ layer differentiation potential. These most-primitive stem cells-those endowed with pluripotent or multipotent differentiation ability and that give rise to other cells more restricted in differentiation, known as tissue-committed stem cells (TCSCs) - are of particular interest. In this review we present the concept supported by accumulating evidence that a population of so-called very small embryonic-like stem cells (VSELs) residing in adult tissues positively impacts the overall survival of mammals, including humans. These unique cells are prevented in vertebrates from premature depletion by decreased sensitivity to growth hormone (GH), insulin (INS), and insulin-like growth factor (IGF) signaling, due to epigenetic changes in paternally imprinted genes that regulate their resistance to these factors. In this context, we can envision nutrient response GH/INS/IGF signaling pathway as a lethal factor for these most primitive stem cells and an important culprit in aging.

An Arf-GAP promotes endocytosis and hyphal growth of Ashbya gossypii.

PubMed

Oscarsson, Therese; Walther, Andrea; Lengeler, Klaus B; Wendland, Jürgen

2017-12-29

The ADP-ribosylation factor (ARF) family of GTPases are highly conserved from yeast to human and regulate vesicle budding. Sec7 domain containing proteins stimulate the guanine nucleotide exchange on Arf proteins, while ARF-GTPase activating proteins stimulate the hydrolysis of GTP. Since vesicle trafficking is important for hyphal growth, we studied the Ashbya gossypii homolog of Saccharomyces cerevisiae ARF3 along with its putative GEF and GTPase-activating protein (GAP) encoded by YEL1 and GTS1, respectively. Deletion of YEL1 had no discernible phenotype and deletion of ARF3 had only a minor defect in vacuolar fusion. In contrast, deletion of GTS1 severely impaired hyphal growth, and mutants showed defects in the maintenance of polarity and the localization of cortical actin patches. The uptake of the lipophilic dye FM4-64 was delayed in gts1 hyphae, indicating a defect in endocytosis. Gts1 has several protein domains, of which the Arf-GAP domain is required for complementation of the gts1 mutant phenotype. GFP-tagged GTS1 under control of its endogenous promoter localized to the plasma membrane but was enriched at hyphal tips and septal sites corresponding to a role in polarized vesicle trafficking. Our results indicate that this ARF-GTPase module plays an important role for filamentous hyphal growth. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The autism associated MET receptor tyrosine kinase engages early neuronal growth mechanism and controls glutamatergic circuits development in the forebrain

PubMed Central

Peng, Yun; Lu, Zhongming; Li, Guohui; Piechowicz, Mariel; Anderson, Miranda; Uddin, Yasin; Wu, Jie; Qiu, Shenfeng

2015-01-01

The human MET gene imparts a replicated risk for autism spectrum disorder (ASD), and is implicated in the structural and functional integrity of brain. MET encodes a receptor tyrosine kinase, MET, which plays a pleiotropic role in embryogenesis and modifies a large number of neurodevelopmental events. Very little is known, however, on how MET signaling engages distinct cellular events to collectively affect brain development in ASD-relevant disease domains. Here, we show that MET protein expression is dynamically regulated and compartmentalized in developing neurons. MET is heavily expressed in neuronal growth cones at early developmental stages and its activation engages small GTPase Cdc42 to promote neuronal growth, dendritic arborization, and spine formation. Genetic ablation of MET signaling in mouse dorsal pallium leads to altered neuronal morphology indicative of early functional maturation. In contrast, prolonged activation of MET represses the formation and functional maturation of glutamatergic synapses. Moreover, manipulating MET signaling levels in vivo in the developing prefrontal projection neurons disrupts the local circuit connectivity made onto these neurons. Therefore, normal time-delimited MET signaling is critical in regulating the timing of neuronal growth, glutamatergic synapse maturation and cortical circuit function. Dysregulated MET signaling may lead to pathological changes in forebrain maturation and connectivity, and thus contribute to the emergence of neurological symptoms associated with ASD. PMID:26728565

Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

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

Zhao, Meng; He, Hong-wei; Sun, Huan-xing

2009-09-18

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, wemore » evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.« less

Kaposi's Sarcoma-Associated Herpesvirus Can Productively Infect Primary Human Keratinocytes and Alter Their Growth Properties

PubMed Central

Cerimele, Francesca; Curreli, Francesca; Ely, Scott; Friedman-Kien, Alvin E.; Cesarman, Ethel; Flore, Ornella

2001-01-01

Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8. At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR. The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR. Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission. PMID:11160746

Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli

PubMed Central

Greene, Sarah E.; Hibbing, Michael E.; Janetka, James; Chen, Swaine L.

2015-01-01

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function. PMID:26126855

Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

PubMed

Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

2006-01-01

Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

The PDZ domain binding motif (PBM) of human T-cell leukemia virus type 1 Tax can be substituted by heterologous PBMs from viral oncoproteins during T-cell transformation.

PubMed

Aoyagi, Tomoya; Takahashi, Masahiko; Higuchi, Masaya; Oie, Masayasu; Tanaka, Yuetsu; Kiyono, Tohru; Aoyagi, Yutaka; Fujii, Masahiro

2010-04-01

Several tumor viruses, such as human T-cell leukemia virus (HTLV), human papilloma virus (HPV), human adenovirus, have high-oncogenic and low-oncogenic subtypes, and such subtype-specific oncogenesis is associated with the PDZ-domain binding motif (PBM) in their transforming proteins. HTLV-1, the causative agent of adult T-cell leukemia, encodes Tax1 with PBM as a transforming protein. The Tax1 PBM was substituted with those from other oncoviruses, and the transforming activity was examined. Tax1 mutants with PBM from either HPV-16 E6 or adenovirus type 9 E4ORF1 are fully active in the transformation of a mouse T-cell line from interleukin-2-dependent growth into independent growth. Interestingly, one such Tax1 PBM mutant had an extra amino acid insertion derived from E6 between PBM and the rest of Tax1, thus suggesting that the amino acid sequences of the peptides between PBM and the rest of Tax1 and the numbers only slightly affect the function of PBM in the transformation. Tax1 and Tax1 PBM mutants interacted with tumor suppressors Dlg1 and Scribble with PDZ-domains. Unlike E6, Tax1 PBM mutants as well as Tax1 did not or minimally induced the degradations of Dlg1 and Scribble, but instead induced their subcellular translocation from the detergent-soluble fraction into the insoluble fraction, thus suggesting that the inactivation mechanism of these tumor suppressor proteins is distinct. The present results suggest that PBMs of high-risk oncoviruses have a common function(s) required for these three tumor viruses to transform cells, which is likely associated with the subtype-specific oncogenesis of these tumor viruses.

Effects of Acute Methamphetamine on Emotional Memory Formation in Humans: Encoding vs Consolidation

PubMed Central

Ballard, Michael E.; Weafer, Jessica; Gallo, David A.; de Wit, Harriet

2015-01-01

Understanding how stimulant drugs affect memory is important for understanding their addictive potential. Here we examined the effects of acute d-methamphetamine (METH), administered either before (encoding phase) or immediately after (consolidation phase) study on memory for emotional and neutral images in healthy humans. Young adult volunteers (N = 60) were randomly assigned to either an encoding group (N = 29) or a consolidation group (N = 31). Across three experimental sessions, they received placebo and two doses of METH (10, 20 mg) either 45 min before (encoding) or immediately after (consolidation) viewing pictures of emotionally positive, neutral, and negative scenes. Memory for the pictures was tested two days later, under drug-free conditions. Half of the sample reported sleep disturbances following the high dose of METH, which affected their memory performance. Therefore, participants were classified as poor sleepers (less than 6 hours; n = 29) or adequate sleepers (6 or more hours; n = 31) prior to analyses. For adequate sleepers, METH (20 mg) administered before encoding significantly improved memory accuracy relative to placebo, especially for emotional (positive and negative), compared to neutral, stimuli. For poor sleepers in the encoding group, METH impaired memory. METH did not affect memory in the consolidation group regardless of sleep quality. These results extend previous findings showing that METH can enhance memory for salient emotional stimuli but only if it is present at the time of study, where it can affect both encoding and consolidation. METH does not appear to facilitate consolidation if administered after encoding. The study also demonstrates the important role of sleep in memory studies. PMID:25679982

The neural representation of social networks.

PubMed

Weaverdyck, Miriam E; Parkinson, Carolyn

2018-05-24

The computational demands associated with navigating large, complexly bonded social groups are thought to have significantly shaped human brain evolution. Yet, research on social network representation and cognitive neuroscience have progressed largely independently. Thus, little is known about how the human brain encodes the structure of the social networks in which it is embedded. This review highlights recent work seeking to bridge this gap in understanding. While the majority of research linking social network analysis and neuroimaging has focused on relating neuroanatomy to social network size, researchers have begun to define the neural architecture that encodes social network structure, cognitive and behavioral consequences of encoding this information, and individual differences in how people represent the structure of their social world. Copyright © 2018 Elsevier Ltd. All rights reserved.

Stimulation of the human medial temporal lobe between learning and recall selectively enhances forgetting

PubMed Central

Merkow, Maxwell B.; Burke, John F.; Ramayya, Ashwin G.; Sharan, Ashwini D.; Sperling, Michael R.; Kahana, Michael J.

2017-01-01

Background Direct electrical stimulation applied to the human medial temporal lobe (MTL) typically disrupts performance on memory tasks, however, the mechanism underlying this effect is not known. Objective To study the effects of MTL stimulation on memory performance Methods We studied the effects of MTL stimulation on memory in five patients undergoing invasive electrocorticographic monitoring during various phases of a memory task (encoding, distractor, recall). Results We found that MTL stimulation disrupted memory performance in a timing-dependent manner; we observed greater forgetting when applying stimulation during the delay between encoding and recall, compared to when it was applied during encoding or recall. Conclusions The results suggest that recall is most dependent on the MTL between learning and retrieval. PMID:28073638

prtH2, Not prtH, Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus▿

PubMed Central

Genay, M.; Sadat, L.; Gagnaire, V.; Lortal, S.

2009-01-01

Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that encode CEPs still remains unclear, rendering it difficult to further control the formation of particular peptides. This study evaluated the diversity of genes that encode CEPs in a collection of strains of L. helveticus isolated from various biotopes, both in terms of the presence or absence of these genes and in terms of nucleotide sequence, and studied their transcription in dairy matrices. After defining three sets of primers for both the prtH and prtH2 genes, we studied the distribution of the genes by using PCR and Southern blotting experiments. The prtH2 gene was ubiquitous in the 29 strains of L. helveticus studied, whereas only 18 of them also exhibited the prtH gene. Sequencing of a 350-bp internal fragment of these genes revealed the existence of intraspecific diversity. Finally, expression of these two CEP-encoding genes was followed during the growth in dairy matrices of two strains, ITG LH77 and CNRZ32, which possess one and two CEP-encoding genes, respectively. Both genes were shown to be expressed by L. helveticus at each stage of growth in milk and at different stages of mini-Swiss-type cheese making and ripening. PMID:19286786

SigmaS controls multiple pathways associated with intracellular multiplication of Legionella pneumophila.

PubMed

Hovel-Miner, Galadriel; Pampou, Sergey; Faucher, Sebastien P; Clarke, Margaret; Morozova, Irina; Morozov, Pavel; Russo, James J; Shuman, Howard A; Kalachikov, Sergey

2009-04-01

Legionella pneumophila is the causative agent of the severe and potentially fatal pneumonia Legionnaires' disease. L. pneumophila is able to replicate within macrophages and protozoa by establishing a replicative compartment in a process that requires the Icm/Dot type IVB secretion system. The signals and regulatory pathways required for Legionella infection and intracellular replication are poorly understood. Mutation of the rpoS gene, which encodes sigma(S), does not affect growth in rich medium but severely decreases L. pneumophila intracellular multiplication within protozoan hosts. To gain insight into the intracellular multiplication defect of an rpoS mutant, we examined its pattern of gene expression during exponential and postexponential growth. We found that sigma(S) affects distinct groups of genes that contribute to Legionella intracellular multiplication. We demonstrate that rpoS mutants have a functional Icm/Dot system yet are defective for the expression of many genes encoding Icm/Dot-translocated substrates. We also show that sigma(S) affects the transcription of the cpxR and pmrA genes, which encode two-component response regulators that directly affect the transcription of Icm/Dot substrates. Our characterization of the L. pneumophila small RNA csrB homologs, rsmY and rsmZ, introduces a link between sigma(S) and the posttranscriptional regulator CsrA. We analyzed the network of sigma(S)-controlled genes by mutational analysis of transcriptional regulators affected by sigma(S). One of these, encoding the L. pneumophila arginine repressor homolog gene, argR, is required for maximal intracellular growth in amoebae. These data show that sigma(S) is a key regulator of multiple pathways required for L. pneumophila intracellular multiplication.

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

PubMed Central

Aoyagi, K; Beyou, A; Moon, K; Fang, L; Ulrich, T

1993-01-01

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat. PMID:8108513

Agrobacterium-mediated transformation of lipomyces

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

Dai, Ziyu; Magnuson, Jon K.; Deng, Shuang

This disclosure provides Agrobacterium-mediated transformation methods for the oil-producing (oleaginous) yeast Lipomyces sp., as well as yeast produced by the method. Such methods utilize Agrobacterium sp. cells that have a T-DNA binary plasmid, wherein the T-DNA binary plasmid comprises a first nucleic acid molecule encoding a first protein and a second nucleic acid molecule encoding a selective marker that permits growth of transformed Lipomyces sp. cells in selective culture media comprising an antibiotic.

Cyclic stretch-induced the cytoskeleton rearrangement and gene expression of cytoskeletal regulators in human periodontal ligament cells.

PubMed

Wu, Yaqin; Zhuang, Jiabao; Zhao, Dan; Zhang, Fuqiang; Ma, Jiayin; Xu, Chun

2017-10-01

This study aimed to explore the mechanism of the stretch-induced cell realignment and cytoskeletal rearrangement by identifying several mechanoresponsive genes related to cytoskeletal regulators in human PDL cells. After the cells were stretched by 1, 10 and 20% strains for 0.5, 1, 2, 4, 6, 12 or 24 h, the changes of the morphology and content of microfilaments were recorded and calculated. Meanwhile, the expression of 84 key genes encoding cytoskeletal regulators after 6 and 24 h stretches with 20% strain was detected by using real-time PCR array. Western blot was applied to identify the protein expression level of several cytoskeletal regulators encoded by these differentially expressed genes. The confocal fluorescent staining results confirmed that stretch-induced realignment of cells and rearrangement of microfilaments. Among the 84 genes screened, one gene was up-regulated while two genes were down-regulated after 6 h stretch. Meanwhile, three genes were up-regulated while two genes were down-regulated after 24 h stretch. These genes displaying differential expression included genes regulating polymerization/depolymerization of microfilaments (CDC42EP2, FNBP1L, NCK2, PIKFYVE, WASL), polymerization/depolymerization of microtubules (STMN1), interacting between microfilaments and microtubules (MACF1), as well as a phosphatase (PPP1R12B). Among the proteins encoded by these genes, the protein expression level of Cdc42 effector protein-2 (encoded by CDC42EP2) and Stathmin-1 (encoded by STMN1) was down-regulated, while the protein expression level of N-WASP (encoded by WASL) was up-regulated. The present study confirmed the cyclic stretch-induced cellular realignment and rearrangement of microfilaments in the human PDL cells and indicated several force-sensitive genes with regard to cytoskeletal regulators.

Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins.

PubMed

Noda, Masafumi; Miyauchi, Rumi; Danshiitsoodol, Narandalai; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

2018-04-01

We have previously shown that the lactic acid bacterium Lactobacillus brevis 174A, isolated from Citrus iyo fruit, produces a bacteriocin designated brevicin 174A, which is comprised of two antibacterial polypeptides (designated brevicins 174A-β and 174A-γ). We have also found a gene cluster, composed of eight open reading frames (ORFs), that contains genes for the biosynthesis of brevicin 174A, self-resistance to its own bacteriocin, and two transcriptional regulatory proteins. Some lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth. Generally, the system consists of a membrane-bound histidine protein kinase (HPK) that senses a specific environmental stimulus and a corresponding response regulator (RR) that mediates the cellular response. We have previously shown that although the HPK- and RR-encoding genes are not found on the brevicin 174A biosynthetic gene cluster in the 174A strain, two putative regulatory genes, designated breD and breG , are in the gene cluster. In the present study, we demonstrate that the expression of brevicin 174A production and self-resistance is positively controlled by two transcriptional regulatory proteins, designated BreD and BreG. BreD is expressed together with BreE as the self-resistance determinant of L. brevis 174A. DNase I footprinting analysis and a promoter assay demonstrated that BreD binds to the breED promoter as a positive autoregulator. The present study also demonstrates that BreG, carrying a transmembrane domain, binds to the common promoter of breB and breC , encoding brevicins 174A-β and 174A-γ, respectively, for positive regulation. IMPORTANCE The problem of the appearance of bacteria that are resistant to practical antibiotics and the increasing demand for safe foods have increased interest in replacing conventional antibiotics with bacteriocin produced by the lactic acid bacteria. This antibacterial substance can inhibit the growth of pathogenic bacteria without side effects on the human body. The bacteriocin that is produced by a Citrus iyo -derived Lactobacillus brevis strain inhibits the growth of pathogenic bacteria such as Listeria monocytogenes , Staphylococcus aureus , and Streptococcus mutans In general, lactic acid bacterial strains have a system to start the production of bacteriocin at an adequate stage of growth, which is called a quorum-sensing system. The system consists of a membrane-bound histidine protein kinase that senses a specific environmental stimulus and a corresponding response regulator that mediates the cellular response. The present study demonstrates that the expression of the genes encoding bacteriocin biosynthesis and the self-resistance determinant is positively controlled by two transcriptional regulatory proteins. Copyright © 2018 American Society for Microbiology.

Induction of protective and therapeutic antitumor immunity by a DNA vaccine with C3d as a molecular adjuvant.

PubMed

Xu, Gui-lian; Zhang, Ke-qin; Guo, Bo; Zhao, Ting-ting; Yang, Fei; Jiang, Man; Wang, Qing-hong; Shang, Yu-hang; Wu, Yu-zhang

2010-10-18

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer therapies is unclear. In this study, we have engineered a DNA vaccine that expresses extracellular region of murine VEGFR-2 (FLK1(265-2493)) and 3 copies of C3d (C3d3), a component of complement as a molecular adjuvant, designed to increase antitumor immunity. VEGFR-2 has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during angiogenesis in the tumor vasculature. Immunization of mice with vector encoding FLK1(265-2493) alone generated only background levels of anti-VEGFR-2 antibodies and slight inhibitory effect on tumor growth. However, the addition of C3d3 to the vaccine construct significantly augmented the anti-VEGFR-2 humoral immune response and inhibited the tumor growth. The antitumor activity induced by vaccination with vector encoding FLK1(265-2493)-C3d3 fusion protein was also demonstrated via growth inhibition of established tumors following passive transfer of immune serum from vaccinated mice. Our results suggest that vaccination with vector encoding FLK1(265-2493) with C3d3 as a molecular adjuvant induces adaptive humoral activity, which is directed against the murine VEGFR-2 and can significantly inhibit tumor growth, and that administration of C3d as a molecular adjuvant to increase antibodies levels to VEGFR-2 may provide an alternative treatment modality for cancer therapies. Copyright © 2010 Elsevier Ltd. All rights reserved.

Expression and function of human hemokinin-1 in human and guinea pig airways.

PubMed

Grassin-Delyle, Stanislas; Naline, Emmanuel; Buenestado, Amparo; Risse, Paul-André; Sage, Edouard; Advenier, Charles; Devillier, Philippe

2010-10-07

Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

FOG: Fighting the Achilles' Heel of Gossip Protocols with Fountain Codes

NASA Astrophysics Data System (ADS)

Champel, Mary-Luc; Kermarrec, Anne-Marie; Le Scouarnec, Nicolas

Gossip protocols are well known to provide reliable and robust dissemination protocols in highly dynamic systems. Yet, they suffer from high redundancy in the last phase of the dissemination. In this paper, we combine fountain codes (rateless erasure-correcting codes) together with gossip protocols for a robust and fast content dissemination in large-scale dynamic systems. The use of fountain enables to eliminate the unnecessary redundancy of gossip protocols. We propose the design of FOG, which fully exploits the first exponential growth phase (where the data is disseminated exponentially fast) of gossip protocols while avoiding the need for the shrinking phase by using fountain codes. FOG voluntarily increases the number of disseminations but limits those disseminations to the exponential growth phase. In addition, FOG creates a split-graph overlay that splits the peers between encoders and forwarders. Forwarder peers become encoders as soon as they have received the whole content. In order to benefit even further and quicker from encoders, FOG biases the dissemination towards the most advanced peers to make them complete earlier.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

PubMed

Schwartzentruber, Jeremy; Buhas, Daniela; Majewski, Jacek; Sasarman, Florin; Papillon-Cavanagh, Simon; Thiffault, Isabelle; Thiffaut, Isabelle; Sheldon, Katherine M; Massicotte, Christine; Patry, Lysanne; Simon, Mariella; Zare, Amir S; McKernan, Kevin J; Michaud, Jacques; Boles, Richard G; Deal, Cheri L; Desilets, Valerie; Shoubridge, Eric A; Samuels, Mark E

2014-11-01

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations. © 2014 WILEY PERIODICALS, INC.

A Novel Hybrid Iron Regulation Network Combines Features from Pathogenic and Nonpathogenic Yeasts

PubMed Central

Gerwien, Franziska; Safyan, Abu; Wisgott, Stephanie; Hille, Fabrice; Kaemmer, Philipp; Linde, Jörg; Brunke, Sascha; Kasper, Lydia

2016-01-01

ABSTRACT Iron is an essential micronutrient for both pathogens and their hosts, which restrict iron availability during infections in an effort to prevent microbial growth. Successful human pathogens like the yeast Candida glabrata have thus developed effective iron acquisition strategies. Their regulation has been investigated well for some pathogenic fungi and in the model organism Saccharomyces cerevisiae, which employs an evolutionarily derived system. Here, we show that C. glabrata uses a regulation network largely consisting of components of the S. cerevisiae regulon but also of elements of other pathogenic fungi. Specifically, similarly to baker’s yeast, Aft1 is the main positive regulator under iron starvation conditions, while Cth2 degrades mRNAs encoding iron-requiring enzymes. However, unlike the case with S. cerevisiae, a Sef1 ortholog is required for full growth under iron limitation conditions, making C. glabrata an evolutionary intermediate to SEF1-dependent fungal pathogens. Therefore, C. glabrata has evolved an iron homeostasis system which seems to be unique within the pathogenic fungi. PMID:27795405

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

PubMed

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-04-01

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Carbon dioxide is a powerful inducer of monokaryotic hyphae and spore development in Cryptococcus gattii and carbonic anhydrase activity is dispensable in this dimorphic transition.

PubMed

Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

2014-01-01

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).

Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

PubMed

Saito, Y; Konnai, S; Yamada, S; Imamura, S; Nishikado, H; Ito, T; Onuma, M; Ohashi, K

2009-08-01

Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

PubMed Central

Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

2014-01-01

Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

Carbon Dioxide is a Powerful Inducer of Monokaryotic Hyphae and Spore Development in Cryptococcus gattii and Carbonic Anhydrase Activity is Dispensable in This Dimorphic Transition

PubMed Central

Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

2014-01-01

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s). PMID:25478697

A tumor suppressor locus within 3p14-p12 mediates rapid cell death of renal cell carcinoma in vivo.

PubMed Central

Sanchez, Y; el-Naggar, A; Pathak, S; Killary, A M

1994-01-01

High frequency loss of alleles and cytogenetic aberrations on the short arm of chromosome 3 have been documented in renal cell carcinoma (RCC). Potentially, three distinct regions on 3p could encode tumor suppressor genes involved in the genesis of this cancer. We report that the introduction of a centric fragment of 3p, encompassing 3p14-q11, into a highly malignant RCC cell line resulted in a dramatic suppression of tumor growth in athymic nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data functionally define a tumor suppressor locus, nonpapillary renal carcinoma-1 (NRC-1), within 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC. Furthermore, we provide functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo. Images PMID:8159756

Discovery of Herpes B Virus-Encoded MicroRNAs▿

PubMed Central

Besecker, Michael I.; Harden, Mallory E.; Li, Guanglin; Wang, Xiu-Jie; Griffiths, Anthony

2009-01-01

Herpes B virus (BV) naturally infects macaque monkeys and is a close relative of herpes simplex virus. BV can zoonotically infect humans to cause a rapidly ascending encephalitis with ∼80% mortality. Therefore, BV is a serious danger to those who come into contact with these monkeys or their tissues and cells. MicroRNAs are regulators of gene expression, and there have been reports of virus-encoded microRNAs. We hypothesize that BV-encoded microRNAs are important for the regulation of viral and cellular genes. Herein, we report the discovery of three herpes B virus-encoded microRNAs. PMID:19144716

Pupil size reflects successful encoding and recall of memory in humans.

PubMed

Kucewicz, Michal T; Dolezal, Jaromir; Kremen, Vaclav; Berry, Brent M; Miller, Laura R; Magee, Abigail L; Fabian, Vratislav; Worrell, Gregory A

2018-03-21

Pupil responses are known to indicate brain processes involved in perception, attention and decision-making. They can provide an accessible biomarker of human memory performance and cognitive states in general. Here we investigated changes in the pupil size during encoding and recall of word lists. Consistent patterns in the pupil response were found across and within distinct phases of the free recall task. The pupil was most constricted in the initial fixation phase and was gradually more dilated through the subsequent encoding, distractor and recall phases of the task, as the word items were maintained in memory. Within the final recall phase, retrieving memory for individual words was associated with pupil dilation in absence of visual stimulation. Words that were successfully recalled showed significant differences in pupil response during their encoding compared to those that were forgotten - the pupil was more constricted before and more dilated after the onset of word presentation. Our results suggest pupil size as a potential biomarker for probing and modulation of memory processing.

Characterization of GM-CSF-inhibitory factor and Uracil DNA glycosylase encoding genes from camel pseudocowpoxvirus.

PubMed

Nagarajan, G; Swami, Shelesh Kumar; Dahiya, Shyam Singh; Narnaware, S D; Mehta, S C; Singh, P K; Singh, Raghvendar; Tuteja, F C; Patil, N V

2015-06-01

The present study describes the PCR amplification of GM-CSF-inhibitory factor (GIF) and Uracil DNA glycosylase (UDG) encoding genes of pseudocowpoxvirus (PCPV) from the Indian Dromedaries (Camelus dromedarius) infected with contagious ecthyma using the primers based on the corresponding gene sequences of human PCPV and reindeer PCPV, respectively. The length of GIF gene of PCPV obtained from camel is 795 bp and due to the addition of one cytosine residue at position 374 and one adenine residue at position 516, the open reading frame (ORF) got altered, resulting in the production of truncated polypeptide. The ORF of UDG encoding gene of camel PCPV is 696 bp encoding a polypeptide of 26.0 kDa. Comparison of amino acid sequence homologies of GIF and UDG of camel PCPV revealed that the camel PCPV is closer to ORFV and PCPV (reference stains of both human and reindeer), respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

PubMed

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Murine c-mpl: a member of the hematopoietic growth factor receptor superfamily that transduces a proliferative signal.

PubMed Central

Skoda, R C; Seldin, D C; Chiang, M K; Peichel, C L; Vogt, T F; Leder, P

1993-01-01

The murine myeloproliferative leukemia virus has previously been shown to contain a fragment of the coding region of the c-mpl gene, a member of the cytokine receptor superfamily. We have isolated cDNA and genomic clones encoding murine c-mpl and localized the c-mpl gene to mouse chromosome 4. Since some members of this superfamily function by transducing a proliferative signal and since the putative ligand of mpl is unknown, we have generated a chimeric receptor to test the functional potential of mpl. The chimera consists of the extracellular domain of the human interleukin-4 receptor and the cytoplasmic domain of mpl. A mouse hematopoietic cell line transfected with this construct proliferates in response to human interleukin-4, thereby demonstrating that the cytoplasmic domain of mpl contains all elements necessary to transmit a growth stimulatory signal. In addition, we show that 25-40% of mpl mRNA found in the spleen corresponds to a novel truncated and potentially soluble isoform of mpl and that both full-length and truncated forms of mpl protein can be immunoprecipitated from lysates of transfected COS cells. Interestingly, however, although the truncated form of the receptor possesses a functional signal sequence and lacks a transmembrane domain, it is not detected in the culture media of transfected cells. Images PMID:8334987

The Y-located gonadoblastoma gene TSPY amplifies its own expression through a positive feedback loop in prostate cancer cells

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

Kido, Tatsuo; Lau, Yun-Fai Chris, E-mail: Chris.Lau@UCSF.edu

2014-03-28

Highlights: • Y-encoded proto-oncoprotein TSPY amplifies its expression level via a positive feedback loop. • TSPY binds to the chromatin/DNA at exon 1 of TSPY gene. • TSPY enhances the gene expression in a TSPY exon 1 sequence dependent manner. • The conserved SET/NAP-domain is essential for TSPY transactivation. • Insights on probable mechanisms on TSPY exacerbation on cancer development in men. - Abstract: The testis-specific protein Y-encoded (TSPY) is a repetitive gene located on the gonadoblastoma region of the Y chromosome, and has been considered to be the putative gene for this oncogenic locus on the male-only chromosome. Itmore » is expressed in spermatogonial cells and spermatocytes in normal human testis, but abundantly in gonadoblastoma, testicular germ cell tumors and a variety of somatic cancers, including melanoma, hepatocellular carcinoma and prostate cancer. Various studies suggest that TSPY accelerates cell proliferation and growth, and promotes tumorigenesis. In this report, we show that TSPY could bind directly to the chromatin/DNA at exon 1 of its own gene, and greatly enhance the transcriptional activities of the endogenous gene in the LNCaP prostate cancer cells. Domain mapping analyses of TSPY have localized the critical and sufficient domain to the SET/NAP-domain. These results suggest that TSPY could efficiently amplify its expression and oncogenic functions through a positive feedback loop, and contribute to the overall tumorigenic processes when it is expressed in various human cancers.« less

SHOX interacts with the chondrogenic transcription factors SOX5 and SOX6 to activate the aggrecan enhancer.

PubMed

Aza-Carmona, Miriam; Shears, Debbie J; Yuste-Checa, Patricia; Barca-Tierno, Verónica; Hisado-Oliva, Alfonso; Belinchón, Alberta; Benito-Sanz, Sara; Rodríguez, J Ignacio; Argente, Jesús; Campos-Barros, Angel; Scambler, Peter J; Heath, Karen E

2011-04-15

SHOX (short stature homeobox-containing gene) encodes a transcription factor implicated in skeletal development. SHOX haploinsufficiency has been demonstrated in Leri-Weill dyschondrosteosis (LWD), a skeletal dysplasia associated with disproportionate short stature, as well as in a variable proportion of cases with idiopathic short stature (ISS). In order to gain insight into the SHOX signalling pathways, we performed a yeast two-hybrid screen to identify SHOX-interacting proteins. Two transcription factors, SOX5 and SOX6, were identified. Co-immunoprecipitation assays confirmed the existence of the SHOX-SOX5 and SHOX-SOX6 interactions in human cells, whereas immunohistochemical studies demonstrated the coexpression of these proteins in 18- and 32-week human fetal growth plates. The SHOX homeodomain and the SOX6 HMG domain were shown to be implicated in the SHOX-SOX6 interaction. Moreover, different SHOX missense mutations, identified in LWD and ISS patients, disrupted this interaction. The physiological importance of these interactions was investigated by studying the effect of SHOX on a transcriptional target of the SOX trio, Agc1, which encodes one of the main components of cartilage, aggrecan. Our results show that SHOX cooperates with SOX5/SOX6 and SOX9 in the activation of the upstream Agc1 enhancer and that SHOX mutations affect this activation. In conclusion, we have identified SOX5 and SOX6 as the first two SHOX-interacting proteins and have shown that this interaction regulates aggrecan expression, an essential factor in chondrogenesis and skeletal development.

The early transcriptional response of human granulocytes to infection with Candida albicans is not essential for killing but reflects cellular communications.

PubMed

Fradin, Chantal; Mavor, Abigail L; Weindl, Günther; Schaller, Martin; Hanke, Karin; Kaufmann, Stefan H E; Mollenkopf, Hans; Hube, Bernhard

2007-03-01

Candida albicans is a polymorphic opportunistic fungus that can cause life-threatening systemic infections following hematogenous dissemination in patients susceptible to nosocomial infection. Neutrophils form part of the innate immune response, which is the first line of defense against microbes and is particularly important in C. albicans infections. To compare the transcriptional response of leukocytes exposed to C. albicans, we investigated the expression of key cytokine genes in polymorphonuclear and mononuclear leukocytes after incubation with C. albicans for 1 h. Isolated mononuclear cells expressed high levels of genes encoding proinflammatory signaling molecules, whereas neutrophils exhibited much lower levels, similar to those observed in whole blood. The global transcriptional profile of neutrophils was examined by using an immunology-biased human microarray to determine whether different morphological forms or the viability of C. albicans altered the transcriptome. Hyphal cells appeared to have the broadest effect, although the most strongly induced genes were regulated independently of morphology or viability. These genes were involved in proinflammatory cell-cell signaling, cell signal transduction, and cell growth. Generally, genes encoding known components of neutrophil granules showed no upregulation at this time point; however, lactoferrin, a well-known candidacidal peptide, was secreted by neutrophils. Addition to inhibitors of RNA or protein de novo synthesis did not influence the killing activity within 30 min. These results support the general notion that neutrophils do not require gene transcription to mount an immediate and direct attack against microbes. However, neutrophils exposed to C. albicans express genes involved in communication with other immune cells.

Human Cytomegalovirus-Encoded miR-US25-1 Aggravates the Oxidised Low Density Lipoprotein-Induced Apoptosis of Endothelial Cells

PubMed Central

Fan, Jianmin; Zhang, Wen; Liu, Qiming

2014-01-01

Human cytomegalovirus (HCMV) infection is linked to the development and severity of the cardiovascular disease atherosclerosis; however, there is little known about the promotion of atherosclerosis. miR-US25-1 is one of HCMV-encoded miRNAs and targets cellular genes that are essential for virus growth to control the life cycle of the virus and host cells. The prominent regulation on cell cycle genes of the miR-US25-1 attracts us to explore its role in the atherosclerosis promotion. It was indicated that miR-US25-1 level was upregulated in subjects or in endothelial cells with HCMV infection; and the miR-US25-1 downregulated the expression of BRCC 3 by targeting the 5′ UTR of BRCC 3. And a miR-US25-1 mimics transfection could reduce the EAhy926 cell viability but did not induce apoptosis in EAhy926 cells. And what is more, miR-US25-1 mimicis transfection deteriorated the ox-LDL-induced apoptosis and aggravated the upregulation of apoptosis-associated molecules by oxidised low density lipoprotein (ox-LDL) in EAhy926 cells. And we have also confirmed the deregulation of BRCC 3 expression by miR-US25-1 by targeting the 5′ UTR of it. Given the vital role of BRCC 3 in DNA damage repairing, we speculated that the targeting inhibition of BRCC 3 by miR-US25-1 may contribute to the aggravation of ox-LDL-promoted apoptosis of endothelial EAhy926 cells. PMID:24895586

Capsule Production and Glucose Metabolism Dictate Fitness during Serratia marcescens Bacteremia

PubMed Central

Anderson, Mark T.; Mitchell, Lindsay A.; Zhao, Lili

2017-01-01

ABSTRACT Serratia marcescens is an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of the Enterobacteriaceae family, the genetic factors that facilitate Serratia proliferation within the mammalian host are less well defined. An in vivo screen of transposon insertion mutants identified 212 S. marcescens fitness genes that contribute to bacterial survival in a murine model of bloodstream infection. Among those identified, 11 genes were located within an 18-gene cluster encoding predicted extracellular polysaccharide biosynthesis proteins. A mutation in the wzx gene contained within this locus conferred a loss of fitness in competition infections with the wild-type strain and a reduction in extracellular uronic acids correlating with capsule loss. A second gene, pgm, encoding a phosphoglucomutase exhibited similar capsule-deficient phenotypes, linking central glucose metabolism with capsule production and fitness of Serratia during mammalian infection. Further evidence of the importance of central metabolism was obtained with a pfkA glycolytic mutant that demonstrated reduced replication in human serum and during murine infection. An MgtB magnesium transporter homolog was also among the fitness factors identified, and an S. marcescens mgtB mutant exhibited decreased growth in defined medium containing low concentrations of magnesium and was outcompeted ~10-fold by wild-type bacteria in mice. Together, these newly identified genes provide a more complete understanding of the specific requirements for S. marcescens survival in the mammalian host and provide a framework for further investigation of the means by which S. marcescens causes opportunistic infections. PMID:28536292

The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes

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

Dyer, K.D.; Handen, J.S.; Rosenberg, H.F.

The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of {beta}-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire {beta}-galactoside bindingmore » site encoded by exon III. We have isolated CLC {beta}-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the {beta}-galactoside ligand. The most likely interpretation of these results suggests the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes. 35 refs., 3 figs.« less

Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

PubMed

Belyanskaya, Svetlana L; Ding, Yun; Callahan, James F; Lazaar, Aili L; Israel, David I

2017-05-04

DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The putative guanine nucleotide exchange factor RicA mediates upstream signaling for growth and development in Aspergillus.

PubMed

Kwon, Nak-Jung; Park, Hee-Soo; Jung, Seunho; Kim, Sun Chang; Yu, Jae-Hyuk

2012-11-01

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.

The Putative Guanine Nucleotide Exchange Factor RicA Mediates Upstream Signaling for Growth and Development in Aspergillus

PubMed Central

Kwon, Nak-Jung; Park, Hee-Soo; Jung, Seunho; Kim, Sun Chang

2012-01-01

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans. PMID:23002107

Growth Cone Localization of the mRNA Encoding the Chromatin Regulator HMGN5 Modulates Neurite Outgrowth

PubMed Central

Moretti, Francesca; Rolando, Chiara; Winker, Moritz; Ivanek, Robert; Rodriguez, Javier; Von Kriegsheim, Alex; Taylor, Verdon; Bustin, Michael

2015-01-01

Neurons exploit local mRNA translation and retrograde transport of transcription factors to regulate gene expression in response to signaling events at distal neuronal ends. Whether epigenetic factors could also be involved in such regulation is not known. We report that the mRNA encoding the high-mobility group N5 (HMGN5) chromatin binding protein localizes to growth cones of both neuron-like cells and of hippocampal neurons, where it has the potential to be translated, and that HMGN5 can be retrogradely transported into the nucleus along neurites. Loss of HMGN5 function induces transcriptional changes and impairs neurite outgrowth, while HMGN5 overexpression induces neurite outgrowth and chromatin decompaction; these effects are dependent on growth cone localization of Hmgn5 mRNA. We suggest that the localization and local translation of transcripts coding for epigenetic factors couple the dynamic neuronal outgrowth process with chromatin regulation in the nucleus. PMID:25825524

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

PubMed Central

Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

2015-01-01

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.

PubMed

Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S

2006-01-01

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.

Computational Analysis Reveals a Key Regulator of Cryptococcal Virulence and Determinant of Host Response

PubMed Central

Gish, Stacey R.; Maier, Ezekiel J.; Haynes, Brian C.; Santiago-Tirado, Felipe H.; Srikanta, Deepa L.; Ma, Cynthia Z.; Li, Lucy X.; Williams, Matthew; Crouch, Erika C.; Khader, Shabaana A.

2016-01-01

ABSTRACT Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes. PMID:27094327

Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones.

PubMed

Kaplan, Ondřej; Zárubová, Jana; Mikulová, Barbora; Filová, Elena; Bártová, Jiřina; Bačáková, Lucie; Brynda, Eduard

2016-01-01

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

PubMed

Dayan, A; Bertrand, R; Beauchemin, M; Chahla, D; Mamo, A; Filion, M; Skup, D; Massie, B; Jolivet, J

1995-11-20

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

Genomic cloning and chromosomal localization of HRY, the human homolog to the Drosophila segmentation gene, hairy

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

Feder, J.N.; Jan, L.Y.; Jan, Y.N.

The Drosophila hairy gene encodes a basic helix- loop-helix protein that functions in at least two steps during Drosophila development: (1) during embryogenesis, when it partakes in the establishment of segments, and (2) during the larval stage, when it functions negatively in determining the pattern of sensory bristles on the adult fly. In the rat, a structurally homologous gene (RHL) behaves as an immediate-early gene in its response to growth factors and can, like that in Drosophila, suppress neuronal differentiation events. Here, the authors report the genomic cloning of the human hairy gene homolog (HRY). The coding region of themore » gene is contained within four exons. The predicted amino acid sequence reveals only four amino acid differences between the human and rat genes. Analysis of the DNA sequence 5[prime] to the coding region reveals a putatitve untranslated exon. To increase the value of the HRY gene as a genetic marker and to assess its potential involvement in genetic disorders, they sublocalized the locus to chromosome 3q28-q29 by fluorescence in situ hybridization. 34 refs., 4 figs., 1 tab.« less

Clinical Consequences of Mutations in Thyroid Hormone Receptor-α1

PubMed Central

van Mullem, Alies A.; Visser, Theo J.; Peeters, Robin P.

2014-01-01

Thyroid hormone (TH) exerts its biological activity via the TH receptors TRα1 and TRβ1/2, which are encoded by the THRA and THRB genes. The first patients with mutations in THRB were identified decades ago. These patients had a clinical syndrome of resistance to TH associated with high serum TH and nonsuppressed thyroid-stimulating hormone levels. Until recently, no patients with mutations in THRA had been identified. In an attempt to predict the clinical phenotype of such patients, different TRα1 mutant mouse models have been generated. These mice have a variable phenotype depending on the location and severity of the mutation. Recently, the first humans with mutations in THRA were identified. Their phenotype consists of relatively low serum T4 and high serum T3 levels (and thus an elevated T3/T4 ratio), growth retardation, delayed mental and bone development, and constipation. While, in retrospect, certain features present in humans can also be found in mouse models, the first humans carrying a defect in TRα1 were not suspected of having a THRA gene mutation initially. The current review focuses on the clinical consequences of TRα1 mutations. PMID:24847461

Increased copper toxicity in Saccharomyces cerevisiae lacking VPS35, a component of the retromer and monogenic Parkinson disease gene in humans.

PubMed

Sowada, Nadine; Stiller, Barbara; Kubisch, Christian

2016-08-05

The Saccharomyces cerevisiae gene VPS35 encodes a component of the retromer complex which is involved in vesicle transport from endosomes to the trans-Golgi network. Yeast and human VPS35 orthologs are highly conserved and mutations in human VPS35 cause an autosomal dominant form of late-onset Parkinson disease (PD). We now show that deletion of VPS35 in yeast (vps35Δ) leads to a dose-dependent growth defect towards copper. This increased sensitivity could be rescued by transformation with yeast wild-type VPS35 but not by the expression of a construct harboring the yeast equivalent (i.e. D686N) of the most commonly identified VPS35-associated PD mutation, p.D620N. In addition, we show that expression of one copy of α-synuclein, which is known to directly interact with copper, leads to a pronounced aggravation of copper toxicity in vps35Δ cells, thereby linking the regulation of copper homeostasis by Vps35p in yeast to one of the key molecules in PD pathophysiology. Copyright © 2016 Elsevier Inc. All rights reserved.

Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system.

PubMed Central

Watson, K L; Konrad, K D; Woods, D F; Bryant, P J

1992-01-01

The tumor suppressor gene lethal(1)aberrant immune response 8 (air8) of Drosophila melanogaster encodes a homolog of the human S6 ribosomal protein. P element insertions that prevent expression of this gene cause overgrowth of the lymph glands (the hematopoietic organs), abnormal blood cell differentiation, and melanotic tumor formation. They also cause delayed development, inhibit growth of most of the larval organs, and lead to larval lethality. Mitotic recombination experiments indicate that the normal S6 gene is required for clone survival in the germ line and imaginal discs. The S6 gene produces a 1.1-kilobase transcript that is abundant throughout development in wild-type animals and in revertants derived from the insertional mutants but is barely detectable in the mutant larvae. cDNAs corresponding to this transcript show a 248-amino acid open reading frame with 75.4% identity and 94.8% similarity to both human and rat S6 ribosomal protein sequences. The results reveal a regulatory function of this ribosomal protein in the hematopoietic system of Drosophila that may be related to its developmentally regulated phosphorylation. Images PMID:1454811

Capsule Production and Glucose Metabolism Dictate Fitness during Serratia marcescens Bacteremia.

PubMed

Anderson, Mark T; Mitchell, Lindsay A; Zhao, Lili; Mobley, Harry L T

2017-05-23

Serratia marcescens is an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of the Enterobacteriaceae family, the genetic factors that facilitate Serratia proliferation within the mammalian host are less well defined. An in vivo screen of transposon insertion mutants identified 212 S. marcescens fitness genes that contribute to bacterial survival in a murine model of bloodstream infection. Among those identified, 11 genes were located within an 18-gene cluster encoding predicted extracellular polysaccharide biosynthesis proteins. A mutation in the wzx gene contained within this locus conferred a loss of fitness in competition infections with the wild-type strain and a reduction in extracellular uronic acids correlating with capsule loss. A second gene, pgm , encoding a phosphoglucomutase exhibited similar capsule-deficient phenotypes, linking central glucose metabolism with capsule production and fitness of Serratia during mammalian infection. Further evidence of the importance of central metabolism was obtained with a pfkA glycolytic mutant that demonstrated reduced replication in human serum and during murine infection. An MgtB magnesium transporter homolog was also among the fitness factors identified, and an S. marcescens mgtB mutant exhibited decreased growth in defined medium containing low concentrations of magnesium and was outcompeted ~10-fold by wild-type bacteria in mice. Together, these newly identified genes provide a more complete understanding of the specific requirements for S. marcescens survival in the mammalian host and provide a framework for further investigation of the means by which S. marcescens causes opportunistic infections. IMPORTANCE Serratia marcescens is a remarkably prolific organism that replicates in diverse environments, including as an opportunistic pathogen in human bacteremia. The genetic requirements for S. marcescens survival in the mammalian bloodstream were defined in this work by transposon insertion sequencing. In total, 212 genes that contribute to bacterial fitness were identified. When sorted via biological function, two of the major fitness categories identified herein were genes encoding capsule polysaccharide biogenesis functions and genes involved in glucose utilization. Further investigation determined that certain glucose metabolism fitness genes are also important for the generation of extracellular polysaccharides. Together, these results identify critical biological processes that allow S. marcescens to colonize the mammalian bloodstream. Copyright © 2017 Anderson et al.

Root gravitropism: a complex response to a simple stimulus?

NASA Technical Reports Server (NTRS)

Rosen, E.; Chen, R.; Masson, P. H.

1999-01-01

Roots avoid depleting their immediate environment of essential nutrients by continuous growth. Root growth is directed by environmental cues, including gravity. Gravity sensing occurs mainly in the columella cells of the root cap. Upon reorientation within the gravity field, the root-cap amyloplasts sediment, generating a physiological signal that promotes the development of a curvature at the root elongation zones. Recent molecular genetic studies in Arabidopsis have allowed the identification of genes that play important roles in root gravitropism. Among them, the ARG1 gene encodes a DnaJ-like protein involved in gravity signal transduction, whereas the AUX1 and AGR1 genes encode proteins involved in polar auxin transport. These studies have important implications for understanding the intra- and inter-cellular signaling processes that underlie root gravitropism.

Protein Analysis with Human Hair

ScienceCinema

Hart, Brad; Anex, Deon; Parker, Glendon

2018-01-16

In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

Protein Analysis with Human Hair

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

Hart, Brad; Anex, Deon; Parker, Glendon

In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.

Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

PubMed Central

2012-01-01

Background In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle. Results Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), assayed under same conditions. This suggested that proteins other than intimin-γ that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion Proteins other than LEE and intimin-γ proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines. PMID:22691138

Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach

PubMed Central

Ragno, Silvia; Romano, Maria; Howell, Steven; Pappin, Darryl J C; Jenner, Peter J; Colston, Michael J

2001-01-01

We investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two-dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up-regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)-8, osteopontin, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), regulated on activation, normal, T-cell expressed and secreted (RANTES), MIP-1β, MIP-3α, myeloid progenitor inhibitory factor-1 (MPIF-1), pulmonary and activation regulated chemokine (PARC), growth regulated gene-β (GRO-β), GRO-γ, MCP-2, I-309, and the T helper 2 (Th2) and eosinophil-attracting chemokine, eotaxin. Other genes involved in cell migration which were up-regulated included the matrix metalloproteinase MMP-9, vascular endothelial growth factor (VEGF) and its receptor Flk-1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule-1 (VCAM-1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL-1β (showing a 433-fold induction), IL-2 and tumour necrosis factor-α (TNF-α), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL-1β was again shown to be strongly up-regulated. The enzyme manganese superoxide dismutase was also found to be strongly up-regulated; this enzyme was found to be macrophage-, rather than M. tuberculosis, derived. The heat-shock protein hsp27 was found to be down-regulated following infection. We also identified a mycobacterial protein, the product of the atpD gene (thought to be involved in the regulation of cytoplasmic pH) in the infected macrophage extracts. PMID:11576227

Stimulation of the human medial temporal lobe between learning and recall selectively enhances forgetting.

PubMed

Merkow, Maxwell B; Burke, John F; Ramayya, Ashwin G; Sharan, Ashwini D; Sperling, Michael R; Kahana, Michael J

Direct electrical stimulation applied to the human medial temporal lobe (MTL) typically disrupts performance on memory tasks, however, the mechanism underlying this effect is not known. To study the effects of MTL stimulation on memory performance. We studied the effects of MTL stimulation on memory in five patients undergoing invasive electrocorticographic monitoring during various phases of a memory task (encoding, distractor, recall). We found that MTL stimulation disrupted memory performance in a timing-dependent manner; we observed greater forgetting when applying stimulation during the delay between encoding and recall, compared to when it was applied during encoding or recall. The results suggest that recall is most dependent on the MTL between learning and retrieval. Copyright © 2017 Elsevier Inc. All rights reserved.

Facilitation of memory encoding in primate hippocampus by a neuroprosthesis that promotes task-specific neural firing

NASA Astrophysics Data System (ADS)

Hampson, Robert E.; Song, Dong; Opris, Ioan; Santos, Lucas M.; Shin, Dae C.; Gerhardt, Greg A.; Marmarelis, Vasilis Z.; Berger, Theodore W.; Deadwyler, Sam A.

2013-12-01

Objective. Memory accuracy is a major problem in human disease and is the primary factor that defines Alzheimer’s, ageing and dementia resulting from impaired hippocampal function in the medial temporal lobe. Development of a hippocampal memory neuroprosthesis that facilitates normal memory encoding in nonhuman primates (NHPs) could provide the basis for improving memory in human disease states. Approach. NHPs trained to perform a short-term delayed match-to-sample (DMS) memory task were examined with multi-neuron recordings from synaptically connected hippocampal cell fields, CA1 and CA3. Recordings were analyzed utilizing a previously developed nonlinear multi-input multi-output (MIMO) neuroprosthetic model, capable of extracting CA3-to-CA1 spatiotemporal firing patterns during DMS performance. Main results. The MIMO model verified that specific CA3-to-CA1 firing patterns were critical for the successful encoding of sample phase information on more difficult DMS trials. This was validated by the delivery of successful MIMO-derived encoding patterns via electrical stimulation to the same CA1 recording locations during the sample phase which facilitated task performance in the subsequent, delayed match phase, on difficult trials that required more precise encoding of sample information. Significance. These findings provide the first successful application of a neuroprosthesis designed to enhance and/or repair memory encoding in primate brain.

Facilitation of Memory Encoding in Primate Hippocampus by a Neuroprosthesis that Promotes Task Specific Neural Firing

PubMed Central

Hampson, Robert E.; Song, Dong; Opris, Ioan; Santos, Lucas M.; Shin, Dae C.; Gerhardt, Greg A.; Marmarelis, Vasilis Z.; Berger, Theodore W.; Deadwyler, Sam A.

2014-01-01

Objective Memory accuracy is a major problem in human disease and is the primary factor that defines Alzheimer’s’, aging and dementia resulting from impaired hippocampal function in medial temporal lobe. Development of a hippocampal memory neuroprosthesis that facilitates normal memory encoding in nonhuman primates (NHPs) could provide the basis for improving memory in human disease states. Approach NHPs trained to perform a short-term delayed match to sample (DMS) memory task were examined with multi-neuron recordings from synaptically connected hippocampal cell fields, CA1 and CA3. Recordings were analyzed utilizing a previously developed nonlinear multi-input multi-output (MIMO) neuroprosthetic model, capable of extracting CA3-to-CA1 spatiotemporal firing patterns during DMS performance. Main Results The MIMO model verified that specific CA3-to-CA1 firing patterns were critical for successful encoding of Sample phase information on more difficult DMS trials. This was validated by delivery of successful MIMO-derived encoding patterns via electrical stimulation to the same CA1 recording locations during the Sample phase which facilitated task performance in the subsequent delayed Match phase on difficult trials that required more precise encoding of Sample information. Significance These findings provide the first successful application of a neuroprosthesis designed to enhance and/or repair memory encoding in primate brain. PMID:24216292

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

PubMed

Fenwick, Aimee L; Kliszczak, Maciej; Cooper, Fay; Murray, Jennie; Sanchez-Pulido, Luis; Twigg, Stephen R F; Goriely, Anne; McGowan, Simon J; Miller, Kerry A; Taylor, Indira B; Logan, Clare; Bozdogan, Sevcan; Danda, Sumita; Dixon, Joanne; Elsayed, Solaf M; Elsobky, Ezzat; Gardham, Alice; Hoffer, Mariette J V; Koopmans, Marije; McDonald-McGinn, Donna M; Santen, Gijs W E; Savarirayan, Ravi; de Silva, Deepthi; Vanakker, Olivier; Wall, Steven A; Wilson, Louise C; Yuregir, Ozge Ozalp; Zackai, Elaine H; Ponting, Chris P; Jackson, Andrew P; Wilkie, Andrew O M; Niedzwiedz, Wojciech; Bicknell, Louise S

2016-07-07

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Influence of the tryptophan-indole-IFNγ axis on human genital Chlamydia trachomatis infection: role of vaginal co-infections.

PubMed

Aiyar, Ashok; Quayle, Alison J; Buckner, Lyndsey R; Sherchand, Shardulendra P; Chang, Theresa L; Zea, Arnold H; Martin, David H; Belland, Robert J

2014-01-01

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.

Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.

PubMed

Hu, Xi-Lian; Zhu, Mu-Yuan; Zhang, Zhi-He; Hou, Rong; Shen, Fu-Jun; Li, Fu-Zhen; Zhang, An-Ju

2006-08-01

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

Complementary DNA characterization and chromosomal localization of a human gene related to the poliovirus receptor-encoding gene.

PubMed

Lopez, M; Eberlé, F; Mattei, M G; Gabert, J; Birg, F; Bardin, F; Maroc, C; Dubreuil, P

1995-04-03

The human poliovirus (PV) receptor (PVR) is a member of the immunoglobulin (Ig) superfamily with unknown cellular function. We have isolated a human PVR-related (PRR) cDNA. The deduced amino acid (aa) sequence of PRR showed, in the extracellular region, 51.7 and 54.3% similarity with human PVR and with the murine PVR homolog, respectively. The cDNA coding sequence is 1.6-kb long and encodes a deduced 57-kDa protein; this protein has a structural organization analogous to that of PVR, that is, one V- and two C-set Ig domains, with a conserved number of aa. Northern blot analysis indicated that a major 5.9-kb transcript is present in all normal human tissues tested. In situ hybridization showed that the PRR gene is located at bands q23-q24 of human chromosome 11.

Subsecond dopamine fluctuations in human striatum encode superposed error signals about actual and counterfactual reward

PubMed Central

Kishida, Kenneth T.; Saez, Ignacio; Lohrenz, Terry; Witcher, Mark R.; Laxton, Adrian W.; Tatter, Stephen B.; White, Jason P.; Ellis, Thomas L.; Phillips, Paul E. M.; Montague, P. Read

2016-01-01

In the mammalian brain, dopamine is a critical neuromodulator whose actions underlie learning, decision-making, and behavioral control. Degeneration of dopamine neurons causes Parkinson’s disease, whereas dysregulation of dopamine signaling is believed to contribute to psychiatric conditions such as schizophrenia, addiction, and depression. Experiments in animal models suggest the hypothesis that dopamine release in human striatum encodes reward prediction errors (RPEs) (the difference between actual and expected outcomes) during ongoing decision-making. Blood oxygen level-dependent (BOLD) imaging experiments in humans support the idea that RPEs are tracked in the striatum; however, BOLD measurements cannot be used to infer the action of any one specific neurotransmitter. We monitored dopamine levels with subsecond temporal resolution in humans (n = 17) with Parkinson’s disease while they executed a sequential decision-making task. Participants placed bets and experienced monetary gains or losses. Dopamine fluctuations in the striatum fail to encode RPEs, as anticipated by a large body of work in model organisms. Instead, subsecond dopamine fluctuations encode an integration of RPEs with counterfactual prediction errors, the latter defined by how much better or worse the experienced outcome could have been. How dopamine fluctuations combine the actual and counterfactual is unknown. One possibility is that this process is the normal behavior of reward processing dopamine neurons, which previously had not been tested by experiments in animal models. Alternatively, this superposition of error terms may result from an additional yet-to-be-identified subclass of dopamine neurons. PMID:26598677

Subsecond dopamine fluctuations in human striatum encode superposed error signals about actual and counterfactual reward.

PubMed

Kishida, Kenneth T; Saez, Ignacio; Lohrenz, Terry; Witcher, Mark R; Laxton, Adrian W; Tatter, Stephen B; White, Jason P; Ellis, Thomas L; Phillips, Paul E M; Montague, P Read

2016-01-05

In the mammalian brain, dopamine is a critical neuromodulator whose actions underlie learning, decision-making, and behavioral control. Degeneration of dopamine neurons causes Parkinson's disease, whereas dysregulation of dopamine signaling is believed to contribute to psychiatric conditions such as schizophrenia, addiction, and depression. Experiments in animal models suggest the hypothesis that dopamine release in human striatum encodes reward prediction errors (RPEs) (the difference between actual and expected outcomes) during ongoing decision-making. Blood oxygen level-dependent (BOLD) imaging experiments in humans support the idea that RPEs are tracked in the striatum; however, BOLD measurements cannot be used to infer the action of any one specific neurotransmitter. We monitored dopamine levels with subsecond temporal resolution in humans (n = 17) with Parkinson's disease while they executed a sequential decision-making task. Participants placed bets and experienced monetary gains or losses. Dopamine fluctuations in the striatum fail to encode RPEs, as anticipated by a large body of work in model organisms. Instead, subsecond dopamine fluctuations encode an integration of RPEs with counterfactual prediction errors, the latter defined by how much better or worse the experienced outcome could have been. How dopamine fluctuations combine the actual and counterfactual is unknown. One possibility is that this process is the normal behavior of reward processing dopamine neurons, which previously had not been tested by experiments in animal models. Alternatively, this superposition of error terms may result from an additional yet-to-be-identified subclass of dopamine neurons.

Polymorphism in Human Cytomegalovirus UL40 Impacts on Recognition of Human Leukocyte Antigen-E (HLA-E) by Natural Killer Cells*

PubMed Central

Heatley, Susan L.; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M. L.; Harpur, Christopher M.; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G.; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C.; Brooks, Andrew G.

2013-01-01

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a “mimic” of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a “polymorphic hot spot” within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors. PMID:23335510

Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells.

PubMed

Heatley, Susan L; Pietra, Gabriella; Lin, Jie; Widjaja, Jacqueline M L; Harpur, Christopher M; Lester, Sue; Rossjohn, Jamie; Szer, Jeff; Schwarer, Anthony; Bradstock, Kenneth; Bardy, Peter G; Mingari, Maria Cristina; Moretta, Lorenzo; Sullivan, Lucy C; Brooks, Andrew G

2013-03-22

Natural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.

Encoding of Natural Sounds at Multiple Spectral and Temporal Resolutions in the Human Auditory Cortex

PubMed Central

Santoro, Roberta; Moerel, Michelle; De Martino, Federico; Goebel, Rainer; Ugurbil, Kamil; Yacoub, Essa; Formisano, Elia

2014-01-01

Functional neuroimaging research provides detailed observations of the response patterns that natural sounds (e.g. human voices and speech, animal cries, environmental sounds) evoke in the human brain. The computational and representational mechanisms underlying these observations, however, remain largely unknown. Here we combine high spatial resolution (3 and 7 Tesla) functional magnetic resonance imaging (fMRI) with computational modeling to reveal how natural sounds are represented in the human brain. We compare competing models of sound representations and select the model that most accurately predicts fMRI response patterns to natural sounds. Our results show that the cortical encoding of natural sounds entails the formation of multiple representations of sound spectrograms with different degrees of spectral and temporal resolution. The cortex derives these multi-resolution representations through frequency-specific neural processing channels and through the combined analysis of the spectral and temporal modulations in the spectrogram. Furthermore, our findings suggest that a spectral-temporal resolution trade-off may govern the modulation tuning of neuronal populations throughout the auditory cortex. Specifically, our fMRI results suggest that neuronal populations in posterior/dorsal auditory regions preferably encode coarse spectral information with high temporal precision. Vice-versa, neuronal populations in anterior/ventral auditory regions preferably encode fine-grained spectral information with low temporal precision. We propose that such a multi-resolution analysis may be crucially relevant for flexible and behaviorally-relevant sound processing and may constitute one of the computational underpinnings of functional specialization in auditory cortex. PMID:24391486

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

PubMed Central

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-01-01

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones. PMID:26593905

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

PubMed

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae.

PubMed

Smith, Christopher P; Thorsness, Peter E

2008-07-01

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.

State and Spectral Properties of Chloride Oscillations in Pollen

PubMed Central

Zonia, Laura; Feijó, José A.

2003-01-01

Pollen tube growth is a dynamic system expressing a number of oscillating circuits. Our recent work identified a new circuit, oscillatory efflux of Cl− anion from the pollen tube apex. Cl− efflux is the first ion signal found to be coupled in phase with growth oscillations. Functional analyses indicate an active role for Cl− flux in pollen tube growth. In this report the dynamical properties of Cl− efflux are examined. Phase space analysis demonstrates that the system trajectory converges on a limit cycle. Fourier analysis reveals that two harmonic frequencies characterize normal growth. Cl− efflux is inhibited by the channel blocker DIDS, is stimulated by hypoosmotic treatment, and is antagonized by the signal encoded in inositol 3,4,5,6-tetrakisphosphate. These perturbations induce transitions of the limit cycle to new metastable states or cause system collapse to a static attractor centered near the origin. These perturbations also transform the spectral profile, inducing subharmonic frequencies, transitions to period doubling and tripling, superharmonic resonance, and chaos. These results indicate that Cl− signals in pollen tubes display features that are characteristic of active oscillators that carry frequency-encoded information. A reaction network of the Cl− oscillator coupled to two nonlinear feedback circuits that may drive pollen tube growth oscillations is considered. PMID:12547818

Integrated transcriptomic and proteomic profiling of white spruce stems during the transition from active growth to dormancy.

PubMed

Galindo González, Leonardo M; El Kayal, Walid; Ju, Chelsea J-T; Allen, Carmen C G; King-Jones, Susanne; Cooke, Janice E K

2012-04-01

In the autumn, stems of woody perennials such as forest trees undergo a transition from active growth to dormancy. We used microarray transcriptomic profiling in combination with a proteomics analysis to elucidate processes that occur during this growth-to-dormancy transition in a conifer, white spruce (Picea glauca[Moench] Voss). Several differentially expressed genes were likely associated with the developmental transition that occurs during growth cessation in the cambial zone and the concomitant completion of cell maturation in vascular tissues. Genes encoding for cell wall and membrane biosynthetic enzymes showed transcript abundance patterns consistent with completion of cell maturation, and also of cell wall and membrane modifications potentially enabling cells to withstand the harsh conditions of winter. Several differentially expressed genes were identified that encoded putative regulators of cambial activity, cell development and of the photoperiodic pathway. Reconfiguration of carbon allocation figured centrally in the tree's overwintering preparations. For example, genes associated with carbon-based defences such as terpenoids were down-regulated, while many genes associated with protein-based defences and other stress mitigation mechanisms were up-regulated. Several of these correspond to proteins that were accumulated during the growth-to-dormancy transition, emphasizing the importance of stress protection in the tree's adaptive response to overwintering. © 2011 Blackwell Publishing Ltd.

Unconscious relational encoding depends on hippocampus

PubMed Central

Duss, Simone B.; Reber, Thomas P.; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M.; Brugger, Peter; Gutbrod, Klemens

2014-01-01

Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age = 45.55 years, standard deviation = 8.74, range = 23–60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. PMID:25273998

Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53.

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

Jiang, H.; Lin, J.; Su, Z.-Z.

The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expressionmore » in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.« less

PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis.

PubMed

Pang, Siew Wai; Lahiri, Chandrajit; Poh, Chit Laa; Tan, Kuan Onn

2018-05-01

Paraneoplastic Ma Family (PNMA) comprises a growing number of family members which share relatively conserved protein sequences encoded by the human genome and is localized to several human chromosomes, including the X-chromosome. Based on sequence analysis, PNMA family members share sequence homology to the Gag protein of LTR retrotransposon, and several family members with aberrant protein expressions have been reported to be closely associated with the human Paraneoplastic Disorder (PND). In addition, gene mutations of specific members of PNMA family are known to be associated with human mental retardation or 3-M syndrome consisting of restrictive post-natal growth or dwarfism, and development of skeletal abnormalities. Other than sequence homology, the physiological function of many members in this family remains unclear. However, several members of this family have been characterized, including cell signalling events mediated by these proteins that are associated with apoptosis, and cancer in different cell types. Furthermore, while certain PNMA family members show restricted gene expression in the human brain and testis, other PNMA family members exhibit broader gene expression or preferential and selective protein interaction profiles, suggesting functional divergence within the family. Functional analysis of some members of this family have identified protein domains that are required for subcellular localization, protein-protein interactions, and cell signalling events which are the focus of this review paper. Copyright © 2018 Elsevier Inc. All rights reserved.

[Human transmissible subacute spongiform encephalopathy].

PubMed

Dormont, D

1994-05-01

Human transmissible spongiform encephalopathies (TSE) are rare chronic subacute degenerative diseases of the central nervous system (CNS) which include Creutzfeldt-Jakob disease (CJD), Kuru, Gerstmann-Sträussler-Scheinker syndrome (GSS), and Fatal Familial Insomnia (FFI). CJD can be either inherited or sporadic. All these diseases are always fatal. Neuropathological features are mainly constituted of neuronal vacuolisation, neuronal death, gliosis with hyperastrocytosis; plaques might be evidenced in kuru and GSS. Neither inflammatory syndrome nor demyelination is detectable. No virus like structure could be identified reproducibly. Human TSE are transmissible to non human primates and rodents. Iatrogenic CJD have been described after tissue grafting (cornea, dura mater), neurosurgery, electrophysiology investigation, and treatment with pituitary derived gonadotrophins and growth hormone. Molecular biochemistry of the CNS investigation revealed that a host encoded protein, the prion protein (PrP), accumulates proportionally to the infectious titer: this abnormality is the only detectable hallmark in TSE. Infectious fractions contain no detectable specific nucleic acid, and are mainly constituted of PrP under an isoform which resists to proteinase K digestion (PrP-res). The PrP gene (PRNP) is located on chromosome 20 in humans. Several mutations of this gene have been described in all inherited TSE (CJD, GSS, and IFF). No treatment is available today. Agents inducing TSE (TSA) are not known: several authors claim that TSA are only constituted of PrP-res; others support the hypothesis of a conventional agent with a specific genetic information.

A Comprehensive Three-Dimensional Cortical Map of Vowel Space

ERIC Educational Resources Information Center

Scharinger, Mathias; Idsardi, William J.; Poe, Samantha

2011-01-01

Mammalian cortex is known to contain various kinds of spatial encoding schemes for sensory information including retinotopic, somatosensory, and tonotopic maps. Tonotopic maps are especially interesting for human speech sound processing because they encode linguistically salient acoustic properties. In this study, we mapped the entire vowel space…

Making Technology Work for Scholarship: Investing in the Data.

ERIC Educational Resources Information Center

Hockey, Susan

This paper examines issues related to how providers and consumers can make the best use of electronic information, focusing on the humanities. Topics include: new technology or old; electronic text and data formats; Standard Generalized Markup Language (SGML); text encoding initiative; encoded archival description (EAD); other applications of…

Molecular Genetic Analysis of Orf Virus: A Poxvirus That Has Adapted to Skin

PubMed Central

Fleming, Stephen B.; Wise, Lyn M.; Mercer, Andrew A.

2015-01-01

Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 132 genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial growth factor genes have been “captured” from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating factor and interleukin-2 appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear factor (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in keratinocytes. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis. PMID:25807056

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Transcription Mapping of the Kaposi’s Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Genome in a Body Cavity-Based Lymphoma Cell Line (BC-1)

PubMed Central

Sarid, Ronit; Flore, Ornella; Bohenzky, Roy A.; Chang, Yuan; Moore, Patrick S.

1998-01-01

Kaposi’s sarcoma-associated herpesvirus (KSHV) gene transcription in the BC-1 cell line (KSHV and Epstein-Barr virus coinfected) was examined by using Northern analysis with DNA probes extending across the viral genome except for a 3-kb unclonable rightmost region. Three broad classes of viral gene transcription have been identified. Class I genes, such as those encoding the v-cyclin, latency-associated nuclear antigen, and v-FLIP, are constitutively transcribed under standard growth conditions, are unaffected by tetradecanoylphorbol acetate (TPA) induction, and presumably represent latent viral transcripts. Class II genes are primarily clustered in nonconserved regions of the genome and include small polyadenylated RNAs (T0.7 and T1.1) as well as most of the virus-encoded cytokines and signal transduction genes. Class II genes are transcribed without TPA treatment but are induced to higher transcription levels by TPA treatment. Class III genes are primarily structural and replication genes that are transcribed only following TPA treatment and are presumably responsible for lytic virion production. These results indicate that BC-1 cells have detectable transcription of a number of KSHV genes, particularly nonconserved genes involved in cellular signal transduction and regulation, during noninduced (latent) virus culture. PMID:9444993

Human motion retrieval from hand-drawn sketch.

PubMed

Chao, Min-Wen; Lin, Chao-Hung; Assa, Jackie; Lee, Tong-Yee

2012-05-01

The rapid growth of motion capture data increases the importance of motion retrieval. The majority of the existing motion retrieval approaches are based on a labor-intensive step in which the user browses and selects a desired query motion clip from the large motion clip database. In this work, a novel sketching interface for defining the query is presented. This simple approach allows users to define the required motion by sketching several motion strokes over a drawn character, which requires less effort and extends the users’ expressiveness. To support the real-time interface, a specialized encoding of the motions and the hand-drawn query is required. Here, we introduce a novel hierarchical encoding scheme based on a set of orthonormal spherical harmonic (SH) basis functions, which provides a compact representation, and avoids the CPU/processing intensive stage of temporal alignment used by previous solutions. Experimental results show that the proposed approach can well retrieve the motions, and is capable of retrieve logically and numerically similar motions, which is superior to previous approaches. The user study shows that the proposed system can be a useful tool to input motion query if the users are familiar with it. Finally, an application of generating a 3D animation from a hand-drawn comics strip is demonstrated.

Diversity, virulence, and antimicrobial resistance of the KPC-producing Klebsiella pneumoniae ST307 clone.

PubMed

Villa, Laura; Feudi, Claudia; Fortini, Daniela; Brisse, Sylvain; Passet, Virginie; Bonura, Celestino; Endimiani, Andrea; Mammina, Caterina; Ocampo, Ana Maria; Jimenez, Judy Natalia; Doumith, Michel; Woodford, Neil; Hopkins, Katie; Carattoli, Alessandra

2017-04-01

The global spread of Klebsiella pneumoniae producing Klebsiella pneumoniae carbapenemase (KPC) has been mainly associated with the dissemination of high-risk clones. In the last decade, hospital outbreaks involving KPC-producing K. pneumoniae have been predominantly attributed to isolates belonging to clonal group (CG) 258. However, results of recent epidemiological analysis indicate that KPC-producing sequence type (ST) 307, is emerging in different parts of the world and is a candidate to become a prevalent high-risk clone in the near future. Here we show that the ST307 genome encodes genetic features that may provide an advantage in adaptation to the hospital environment and the human host. Sequence analysis revealed novel plasmid-located virulence factors, including a cluster for glycogen synthesis. Glycogen production is considered to be one of the possible adaptive responses to long-term survival and growth in environments outside the host. Chromosomally-encoded virulence traits in the clone comprised fimbriae, an integrative conjugative element carrying the yersiniabactin siderophore, and two different capsular loci. Compared with the ST258 clone, capsulated ST307 isolates showed higher resistance to complement-mediated killing. The acquired genetic features identified in the genome of this new emerging clone may contribute to increased persistence of ST307 in the hospital environment and shed light on its potential epidemiological success.

Investigation of alterations in multifractality in optical coherence tomographic images of in vivo human retina

NASA Astrophysics Data System (ADS)

Das, Nandan Kumar; Mukhopadhyay, Sabyasachi; Ghosh, Nirmalya; Chhablani, Jay; Richhariya, Ashutosh; Divakar Rao, Kompalli; Sahoo, Naba Kishore

2016-09-01

Optical coherence tomography (OCT) enables us to monitor alterations in the thickness of the retinal layer as disease progresses in the human retina. However, subtle morphological changes in the retinal layers due to early disease progression often may not lead to detectable alterations in the thickness. OCT images encode depth-dependent backscattered intensity distribution arising due to the depth distributions of the refractive index from tissue microstructures. Here, such depth-resolved refractive index variations of different retinal layers were analyzed using multifractal detrended fluctuation analysis, a special class of multiresolution analysis tools. The analysis extracted and quantified microstructural multifractal information encoded in normal as well as diseased human retinal OCT images acquired in vivo. Interestingly, different layers of the retina exhibited different degrees of multifractality in a particular retina, and the individual layers displayed consistent multifractal trends in healthy retinas of different human subjects. In the retinal layers of diabetic macular edema (DME) subjects, the change in multifractality manifested prominently near the boundary of the DME as compared to the normal retinal layers. The demonstrated ability to quantify depth-resolved information on multifractality encoded in OCT images appears promising for the early diagnosis of diseases of the human eye, which may also prove useful for detecting other types of tissue abnormalities from OCT images.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

PubMed

Gao, J; Naglich, J G; Laidlaw, J; Whaley, J M; Seizinger, B R; Kley, N

1995-02-15

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

PubMed Central

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T.; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-01-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/). PMID:12634390

In silico pattern-based analysis of the human cytomegalovirus genome.

PubMed

Rigoutsos, Isidore; Novotny, Jiri; Huynh, Tien; Chin-Bow, Stephen T; Parida, Laxmi; Platt, Daniel; Coleman, David; Shenk, Thomas

2003-04-01

More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/).

Potential of Gene Therapy for the Treatment of Pituitary Tumors

PubMed Central

Goya, R G.; Sarkar, D.K.; Brown, O.A.; Hereñú, C.B.

2010-01-01

Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy—the transfer of genetic material for therapeutic purposes—has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined. PMID:15032616

Feature-Selective Attentional Modulations in Human Frontoparietal Cortex.

PubMed

Ester, Edward F; Sutterer, David W; Serences, John T; Awh, Edward

2016-08-03

Control over visual selection has long been framed in terms of a dichotomy between "source" and "site," where top-down feedback signals originating in frontoparietal cortical areas modulate or bias sensory processing in posterior visual areas. This distinction is motivated in part by observations that frontoparietal cortical areas encode task-level variables (e.g., what stimulus is currently relevant or what motor outputs are appropriate), while posterior sensory areas encode continuous or analog feature representations. Here, we present evidence that challenges this distinction. We used fMRI, a roving searchlight analysis, and an inverted encoding model to examine representations of an elementary feature property (orientation) across the entire human cortical sheet while participants attended either the orientation or luminance of a peripheral grating. Orientation-selective representations were present in a multitude of visual, parietal, and prefrontal cortical areas, including portions of the medial occipital cortex, the lateral parietal cortex, and the superior precentral sulcus (thought to contain the human homolog of the macaque frontal eye fields). Additionally, representations in many-but not all-of these regions were stronger when participants were instructed to attend orientation relative to luminance. Collectively, these findings challenge models that posit a strict segregation between sources and sites of attentional control on the basis of representational properties by demonstrating that simple feature values are encoded by cortical regions throughout the visual processing hierarchy, and that representations in many of these areas are modulated by attention. Influential models of visual attention posit a distinction between top-down control and bottom-up sensory processing networks. These models are motivated in part by demonstrations showing that frontoparietal cortical areas associated with top-down control represent abstract or categorical stimulus information, while visual areas encode parametric feature information. Here, we show that multivariate activity in human visual, parietal, and frontal cortical areas encode representations of a simple feature property (orientation). Moreover, representations in several (though not all) of these areas were modulated by feature-based attention in a similar fashion. These results provide an important challenge to models that posit dissociable top-down control and sensory processing networks on the basis of representational properties. Copyright © 2016 the authors 0270-6474/16/368188-12$15.00/0.

Upconversion Nanoparticles-Encoded Hydrogel Microbeads-Based Multiplexed Protein Detection

NASA Astrophysics Data System (ADS)

Shikha, Swati; Zheng, Xiang; Zhang, Yong

2018-06-01

Fluorescently encoded microbeads are in demand for multiplexed applications in different fields. Compared to organic dye-based commercially available Luminex's xMAP technology, upconversion nanoparticles (UCNPs) are better alternatives due to their large anti-Stokes shift, photostability, nil background, and single wavelength excitation. Here, we developed a new multiplexed detection system using UCNPs for encoding poly(ethylene glycol) diacrylate (PEGDA) microbeads as well as for labeling reporter antibody. However, to prepare UCNPs-encoded microbeads, currently used swelling-based encapsulation leads to non-uniformity, which is undesirable for fluorescence-based multiplexing. Hence, we utilized droplet microfluidics to obtain encoded microbeads of uniform size, shape, and UCNPs distribution inside. Additionally, PEGDA microbeads lack functionality for probe antibodies conjugation on their surface. Methods to functionalize the surface of PEGDA microbeads (acrylic acid incorporation, polydopamine coating) reported thus far quench the fluorescence of UCNPs. Here, PEGDA microbeads surface was coated with silica followed by carboxyl modification without compromising the fluorescence intensity of UCNPs. In this study, droplet microfluidics-assisted UCNPs-encoded microbeads of uniform shape, size, and fluorescence were prepared. Multiple color codes were generated by mixing UCNPs emitting red and green colors at different ratios prior to encapsulation. UCNPs emitting blue color were used to label the reporter antibody. Probe antibodies were covalently immobilized on red UCNPs-encoded microbeads for specific capture of human serum albumin (HSA) as a model protein. The system was also demonstrated for multiplexed detection of both human C-reactive protein (hCRP) and HSA protein by immobilizing anti-hCRP antibodies on green UCNPs.

Genome Sequence of the Obligate Gammaproteobacterial Methanotroph Methylomicrobium album strain BG8

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

Kits, K. Dimitri; Kalyuzhnaya, Marina G.; Klotz, Martin G

2013-01-01

The complete genome sequence of Methylomicrobium album BG8, a methane-oxidizing gammaproteobacterium isolated from freshwater, is reported. Aside from conserved inventory for growth on single-carbon compounds, M. album BG8 encodes a range of inventory for additional carbon and nitrogen transformations, but no genes for growth on multi-carbon substrates or for N-fixation.

Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar

Treesearch

Steven L. Voelker; Barbara Lachenbruch; Frederick C. Meinzer; Michael Jourdes; Chanyoung Ki; Ann M. Patten; Laurence B. Davin; Norman G. Lewis; Gerald A. Tuskan; Lee Gunter; Stephen R. Decker; Michael J. Selig; Robert Sykes; Michael E. Himmel; Peter Kitin; Olga Shevchenko; Steven H. Strauss

2010-01-01

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula...

Parietal and superior frontal visuospatial maps activated by pointing and saccades

PubMed Central

Hagler, D.J.; Riecke, L.; Sereno, M.I.

2009-01-01

A recent study from our laboratory demonstrated that parietal cortex contains a map of visual space related to saccades and spatial attention and identified this area as the likely human homologue of the lateral intraparietal (LIP). A human homologue for the parietal reach region (PRR), thought to preferentially encode planned hand movements, has also been recently proposed. Both of these areas, originally identified in the macaque monkey, have been shown to encode space with eye-centered coordinates. Functional magnetic resonance imaging (fMRI) of humans was used to test the hypothesis that the putative human PRR contains a retinotopic map recruited by finger pointing but not saccades and to test more generally for differences in the visuospatial maps recruited by pointing and saccades. We identified multiple maps in both posterior parietal cortex and superior frontal cortex recruited for eye and hand movements, including maps not observed in previous mapping studies. Pointing and saccade maps were generally consistent within single subjects. We have developed new group analysis methods for phase-encoded data, which revealed subtle differences between pointing and saccades, including hemispheric asymmetries, but we did not find evidence of pointing-specific maps of visual space. PMID:17376706

Unusual varieties and duplication of Rig-I like receptors encoded in the marine mollusk, Crassostrea gigas

NASA Astrophysics Data System (ADS)

Tian, Z. H.; Jiao, C. Z.

2017-07-01

RIG-I like receptors (RLRs) play key roles in sensing non-self nucleic acids in cytoplasm and trigger antiviral innate immune response in vertebrates and human body. Here we carried out in silico analysis to identify and investigate the putative RLRs encoded in the genome of marine mollusk, Crassostrea gigas (cgRLRs), an invertebrate species. We found the unusual duplication and varieties on domain architecture of putative cgRLRs encoded in the genome of C. gigas. Three putative cgRLRs (accessions numbers are EKC24603, EKC31344.1 and EKC38304.1 on GenBank), have the similar domain architecture with that of human RIG-I or MDA5, and one protein (EKC34573.1) with that of human LGP2; The fifth putative cgRLRs (EKC38303.1) is somewhat similar with human RIG-I/MDA5 except that it has only one caspase activation and recruitment domain (CARD) in its N-terminal. Other nine proteins were identified to be partialy similar with RLRs while with the incomplete sequences, which maybe reflect the events of partial duplication of cgRLRs genes occurred in the oyster genome.

Deregulated Methionine Adenosyltransferase α1, c-Myc and Maf Proteins Interplay Promotes Cholangiocarcinoma Growth in Mice and Humans

PubMed Central

Yang, Heping; Liu, Ting; Wang, Jiaohong; Li, Tony W.H.; Fan, Wei; Peng, Hui; Krishnan, Anuradha; Gores, Gregory J.; Mato, Jose M.; Lu, Shelly C.

2016-01-01

We reported c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice. We also showed induction of Maf proteins (MafG and c-Maf) contributed to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration was protective. Here we determined whether there is interplay between c-Myc, Maf proteins and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms and functional outcomes. We found c-Myc, MATα1 (encoded by MAT1A), MafG and c-Maf interact with each other directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver but this switches to c-Maf, MafG and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG and c-Maf, but it negatively regulates MAT1A. MATα1 represses whereas c-Myc, MafG and c-Maf enhance E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. Conclusion We have uncovered a novel interplay between MATα1, c-Myc and Maf proteins and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. PMID:26969892

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

PubMed

Bachvarov, D R; Hess, J F; Menke, J G; Larrivée, J F; Marceau, F

1996-05-01

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.