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I Kola

Publications and source records attributed to I Kola.

At least 73 records · Page 4Linked to original sources
PubMed1997

Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

3T3 Cells↗
PubMed1997

Response of a primary human fibroblast cell line to H2O2: senescence-like growth arrest or apoptosis?

Hydrogen peroxide has been shown to induce either apoptosis or features of senescence in different cultured cell lines. We now show that both processes can be induced in the same culture of primary human diploid fibroblasts and that the outcome of apoptosis or the senescence-like phenotype is determined by the H2O2 concentrations. At 50 and 100 microM, H2O2 predominantly induced the senescence-like state, characterized by a reduced rate of proliferation, an increased number of cells in G0-G1, typically enlarged and flattened morphology, and increased CIP1 and fibronectin expression. At 300 and 400 microM, H2O2 mainly triggered apoptosis. At the intermediate 200 microM H2O2, features of both senescence and apoptosis were observed in the same culture. Thus, the higher the H2O2 concentration, the higher the proportion of cells undergoing apoptosis, suggesting a key role of the level of damage in the choice of a cell population to enter apoptosis and/or the senescence-like state. Before the induction of one or the other process, cells entered a transient "shock state" characterized by a typical morphological change, cell cycle arrest in G0-G1, and the induction of CIP1 and BCL-2.

Apoptosis↗
PubMed1996

Endogenous IFN-alpha beta suppresses colony-stimulating factor (CSF)-1-stimulated macrophage DNA synthesis and mediates inhibitory effects of lipopolysaccharide and TNF-alpha.

Murine bone marrow-derived macrophages (BMM) are widely used as a suitable model to study the proliferative response to macrophage-CSF or CSF-1. We report here that the amount of DNA synthesis observed in BMM cultures in response to CSF-1 can be masked quite significantly by low levels of IFN-alpha beta produced in the cultures. It was found that Ab to IFN-alpha beta could enhance the proliferative response in CSF-treated BMM that were able to respond to endogenous IFN-alpha beta; however, BMM from mice lacking a component of the type I IFN receptor did not show any enhancement of CSF-1-dependent DNA synthesis on addition of the Ab. While DNA synthesis in CSF-1-stimulated BMM from normal mice was also very sensitive to the inhibitory actions of very low concentrations of added IFN-alpha beta, DNA synthesis in BMM from the "knockout" mice was not, indicating that the type I IFN receptor component containing the null mutation was essential for signal transduction. Previously it was shown that bacterial LPS, TNF-alpha, IFN-gamma, and cAMP could all inhibit CSF-1-stimulated BMM DNA synthesis and proliferation. Using the combined approach of blocking IFN-alpha beta Ab and the IFN receptor "knockout" mice, it was found here that the growth-inhibitory effects of LPS and TNF-alpha are due, to a significant extent, to endogenous IFN-alpha beta, whereas those of IFN-gamma and cAMP occur by a different mechanism. it is proposed that the type I IFN receptor (IFNAR 1) "knockout" mice may be useful in delineating some of the in vivo actions of CSF-1, LPS, TNF-alpha, and possibly other agents.

Animals↗
PubMed1996

ETS1 and ETS2 in p53 regulation: spatial separation of ETS binding sites (EBS) modulate protein: DNA interaction.

p53 is an extensively studied tumor suppressor gene implicated in the genesis of a large number of varied tumors. However, the pathways of regulation for the wild-type p53 gene and its product are as yet unknown. In situ hybridization analyses of ETS1 and ETS2 expression during mouse embryogenesis, have shown a pattern similar to that of p53 gene expression. Significantly, we have identified several ETS-binding sites (EBS) in the promoter regions of the human and mouse p53 genes. In the human promoter two of these EBS are present in the form of a palindrome, with the two EBS cores being separated by four nucleotides. This report shows that the EBS palindrome of the human p53 promoter has a high affinity for ETS1 and ETS2 and that such binding interaction intracellularly is able to activate the transcription of a CAT reporter gene by 5-10-fold using COS cells. To investigate whether the spacing between the two EBS cores influences the DNA binding activity, we synthesized oligonucleotides with increasing distances (4,12,16, and 20 bases respectively) between the two EBS cores of the palindrome. We observed an inverse correlation between an increasing distance in the two EBS cores of the palindrome and the ETS1 and ETS2 DNA binding activity respectively. Interestingly, optimal DNA binding activity was observed when the distance between the two EBS cores was four bases, identical to that which occurs in the natural promoter. Furthermore we show that the p53 mRNA is expressed at higher levels in NIH3T3 cells overexpressing ETS2 gene product, suggesting that the ETS2 transcription factor is a likely candidate for regulating the expression of p53 in vivo.

3T3 Cells↗
PubMed1996

Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice.

Expression of Ets2, a proto-oncogene and transcription factor, occurs in a variety of cell types. During murine development it is highly expressed in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Ets2 is located on human chromosome 21 (ref. 8) and is overexpressed in Down's syndrome (trisomy 21). Here we generate transgenic mice to investigate the consequences of overexpression of Ets2. We find that mice with less than 2-fold Ets2 overexpression in particular organs develop neurocranial, viscerocranial and cervical skeletal abnormalities. These abnormalities have similarities with the skeletal anomalies found in trisomy-16 mice and humans with Down's syndrome, in which the gene dosage of Ets2 is increased. Our results indicate that Ets2 has a role in skeletal development and implicate the overexpression of Ets2 in the genesis of some skeletal abnormalities that occur in Down's syndrome.

Abnormalities, Multiple↗
PubMed1996

Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.

Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones.

Animals↗
PubMed1996

Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes.

Mouse oocytes have proven useful in experiments aimed at studying gene function. They have been used to analyze the gain-of-function acquired after microinjection of RNA transcribed in vitro from specific gene constructs, and for establishing loss-of-function mutation obtained by injecting in vitro transcribed antisense RNA and/or synthetic oligonucleotides. This article presents protocols utilized in these studies. Specifically, the acquisition of mouse oocytes and/or embryos, the genesis of the necessary DNA and/or RNA to be used, and procedures for microinjection.

Animals↗
PubMed1996

Binding of interferon-alpha and -beta to a component of the human type I interferon receptor expressed in simian cells.

The type I interferons (IFNs) are a family of homologous cytokines that compete for receptor binding. Past experiments with a cloned human IFN-alpha receptor component (designated herein as HuIFNAR-1) transfected into different cell backgrounds have given contradictory results in terms of binding and signalling after exposure of cells to different human type I IFNs. In order to investigate the binding specificity of human type I IFN subtypes to HuIFNAR-1, a cDNA encoding HuIFNAR-1 was transfected into simian COS cells. HuIFNAR-1 expression in COS cells, which was confirmed by Northern blot analysis, resulted in increased binding of 125I-labelled HuIFN-alpha 2 and -beta. These data support the participation of this receptor component in ligand binding, probably in association with other receptor components.

Animals↗
PubMed1996

Analysis of heat shock protein 70 in human chromosome 21 containing hybrids.

An Hsp70 gene, HspA3, has been given provisional assignment to chromosome 21 based on the heat induced expression of a 70 kDa protein in a Chinese hamster ovary-human hybrid cell line. This assignment has not been supported by hybridization data or by cloning of the gene. The aim of the current study is to clarify this localization. We have analysed a series of Chinese hamster ovary-human hybrid lines containing human chromosome 21 by two-dimensional gel electrophoresis and Western blot analysis with anti-Hsp70 antibodies. Only one of these, hybrid 72532-X6, was found to contain an additional Hsp70 protein. Three other hybrid cell lines spanning different parts of chromosome 21 did not contain any additional Hsp70 proteins. The Hsp70 protein in 72532-X6 is constitutively expressed and is not heat inducible. Southern blot analysis indicates that this protein is not the major human cognate protein, Hsp73 (Hsp70-8), but may be one of the other cognate Hsp70s. Our data indicate that hybrid cell lines containing human chromosome 21 do not express a human Hsp70, as has been reported previously. The recent report that the hybrid cell line used to make this claim also contains other human chromosome fragments leads us to conclude that an Hsp70 gene is not localized on human chromosome.

Animals↗
PubMed1996

Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide.

Although reactive oxygen species have been proposed to play a major role in the aging process, the exact molecular mechanisms remain elusive. In this study we investigate the effects of a perturbation in the ratio of Cu/Zn-superoxide dismutase activity (Sod1 dismutases .O2-to H2O2) to glutathione peroxidase activity (Gpx1 catalyses H2O2 conversion to H2O) on cell growth and development. Our data demonstrate that Sod1 transfected cell lines that have an elevation in the ratio of Sod1 activity to Gpx1 activity produce higher levels of H2O2 and exhibit well characterised markers of cellular senescence viz. slower proliferation and altered morphology. On the contrary, Sod1 transfected cell lines that have an unaltered ratio in the activity of these two enzymes, have unaltered levels of H2O2 and fail to show characteristics of senescence. Furthermore, fibroblasts established from individuals with Down syndrome have an increase in the ratio of Sod1 to Gpx1 activity compared with corresponding controls and senesce earlier. Interestingly, cells treated with H2O2 also show features of senescence and/or senesce earlier. We also show that Cip1 mRNA levels are elevated in Down syndrome cells, Sod1 transfectants with an altered Sod1 to Gpx1 activity ratio and those treated with H2O2, thus suggesting that the slow proliferation may be mediated by Cip1. Furthermore, our data demonstrate that Cip1 mRNA levels are induced by exposure of cells to H2O2. These data give valuable insight into possible molecular mechanisms that contribute tribute to cellular senescence and may be useful in the evolution of therapeutic strategies for aging.

Base Sequence↗
PubMed1996

A hitchhiker's guide to the human Hsp70 family.

The human Hsp70 family encompasses at least 11 genes which encode a group of highly related proteins. These proteins include both cognate and highly inducible members, at least some of which act as molecular chaperones. The location of cognate Hsp70s within all the major subcellular compartments is an indication of the importance of these proteins. The expression of several inducible Hsp70 genes is also an indication of the importance of these proteins in the stress response. The existence of multiple genes and protein isoforms has created confusion in the identification and naming of particular family members. We have compiled, from the literature, a list of genes and genetic loci and produced a two-dimensional protein map of the known human Hsp70 family members. This will enable researchers in the field to quickly and reliably identify human Hsp70s. We have also devised a more rational nomenclature for these genes and gene products which, subject to general acceptance, could be extended to Hsp70 families from other species.

Blotting, Western↗
PubMed1995

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Animals↗
PubMed1995

ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.

Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.

Base Sequence↗
PubMed1995

Localization of the gene encoding the human heat shock cognate protein, HSP73, to chromosome 11.

The heat shock cognate protein HSP73 (or HSC70) is a member of the HSP70 multigene family. This protein has several functions, including binding to nascent polypeptides to facilitate correct folding and the uncoating of clathrin-coated vesicles. Analysis of somatic cell hybrids by two-dimensional protein gel electrophoresis revealed the presence of a 73-kDa protein in two hybrids containing human chromosomes 5, 6, 9, and 11 in common. Using Western blot analysis, we demonstrate that this protein is a member of the HSP70 family and, by Southern blot analysis, that the HSP73 gene is located on human chromosome 11. Fluorescence in situ hybridization further localized HSP73 to the region 11q23.3-q25. This region is involved in a number of genetic rearrangements and is associated with several well-characterized tumours.

Animals↗
PubMed1995

Changes in the levels of enzymes which modulate the antioxidant balance occur during aging and correlate with cellular damage.

Oxidative metabolism produces a flux of superoxide anions that must be removed from the cellular environment if the cell is to survive. The levels of antioxidant enzyme involved in the elimination of superoxide anions and/or hydrogen peroxide were investigated in an attempt to correlate any changes in the levels of these enzymes during aging with changes in free radical mediated cellular damage. Cu/Zn superoxide dismutase (Sod1), glutathione peroxidase (Gpx1) and catalase levels were measured in a number of organs during murine aging. Sod1 enzyme activity rose during aging in all organs studied, while the levels of both Gpx1 and catalase showed organ specific profiles. Both organs in which lipid peroxidation damage (which was used as a marker of free radical mediated damage) increased with age, namely the brain and small intestine, also showed a significant increase in the ratio of Sod1 to Gpx1 enzyme activity. In organs where either the ratio of Sod1/Gpx1 activity or Sod1/catalase levels (in the lung only) ratios were maintained during aging, no increased lipid peroxidation damage was detected. In the lung where Sod1/Gpx1 ratio did increase, Sod1/catalase remained constant and this was able to provide protection during aging. Thus our data shows that alterations in the balance between first and second steps of the antioxidant pathway correlate with cellular damage, and that this may contribute to the aging changes seen in some organs.

Age Factors↗
PubMed1995

Human ERG is a proto-oncogene with mitogenic and transforming activity.

The ETS related gene, ERG, is one of 20 or more genes belonging to the ETS family of transcription factors. Translocation of the ERG gene t(21;22) results in the chimeric fusion transcript seen in approximately 10% of Ewings sarcomas. In addition, recent studies have shown that a reciprocal translocation t(21;16) of ERG gives rise to two aberrant transcripts seen in some forms of acute myeloid leukaemia. In vitro studies have linked the up regulation of ERG expression with stromal cell independence in erythroleukemic clones and shown that the ERG related genes ETS1 and ETS2 have a mitogenic and transforming activity when overexpressed in NIH3T3 cells. Interestingly ERGB/FLI-1, which is also involved in Ewings sarcoma translocations and shares a very high sequence identify with ERG has been reported to be unable to transform NIH3T3 cells. In this study we investigate the effects of overexpression of ERG on cell proliferation, factor dependence, growth in soft agar and tumorigenesis in nude mice. An ERG expression construct with the human ERG2 cDNA driven by the sheep metallothionein la promoter (sMTERG) was transfected into NIH3T3 cells. Clonal cell lines overexpressing ERG were established. The cell lines became morphologically altered, grew in low serum and serum free media and gave rise to colonies in soft agar suspension. Furthermore, we demonstrate that after subcutaneous injection these clones grow as solid tumors in nude mice. These data demonstrate that c-ERG is a proto-oncogene capable of transforming NIH3T3 cells. Therefore, overexpression or inappropriate expression of ERG may contribute to oncogenesis.

3T3 Cells↗
PubMed1995

A model for understanding gene regulation during spermatogenesis: the mouse testis Pdha-2 promoter.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. How these genes are regulated during spermatogenesis is poorly understood. However, the elucidation of these mechanisms has significant implications for both medicine and the primary livestock industry. The delineation of this process is of particular interest and, in this study, Pdha-2, a gene which codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex, has been used as a model. This review focuses on current knowledge about its expression and regulation during spermatogenesis.

Animals↗