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T Evans

Publications and source records attributed to T Evans.

At least 91 records · Page 5Linked to original sources
PubMed1997

Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter.

Nitric oxide production in a variety of inflammatory conditions is dependent on the synthesis of the enzyme, inducible nitric-oxide synthase (iNOS). The gene for this enzyme is regulated by a number of inflammatory cytokines, including interferon-gamma. Transcriptional activation of the gene is dependent on the interferon-gamma-induced transcription factor, interferon regulatory factor-1 (IRF-1). Using a 99-base pair segment of the iNOS gene promoter encompassing nucleotides -979 to -881, a region essential for gene activation by cytokines, we show that with increasing concentrations of added IRF-1, a monomeric then a dimeric complex form. Molecular footprinting analysis shows that the factor binds initially to a canonical IRF-1 site as a monomer. The region of binding is then extended both in a 5' and 3' direction on formation of the dimeric complex, with additional contacts in the minor groove of DNA. Binding of the second molecule of IRF-1 is dependent on the presence of the initial bound protein. Sequential binding of IRF-1 to form a dimeric complex has not been described previously, and we show that formation of this dimeric complex is essential for full activation of the iNOS gene by cytokines in vascular smooth muscle cells.

Animals↗
PubMed1997

Developmental biology of hematopoiesis.

Hematopoietic stem cells are at the top of a hierarchy that regulates the generation of a vast repertoire of blood cells during the lifetime of a vertebrate. Recent experiments, using a vast variety of embryonic systems, shed new light on the origin of stem cells and the genes that function to regulate and maintain hematopoietic differentiation programs. Two distinct populations of stem cells develop--derived initially from transient, extraembryonic source and later from a stable, intraembryonic source; it is possible that both are generated from a pro-HSC able to respond differentially to local inductions. The initial blood cells develop from ventral mesoderm. The blood-forming region develops as a result of signaling from specific, secreted, embryonic growth factors, including the bone morphogenetic proteins. Stem cells give rise to progenitors that are restricted progressively in their ability to contribute to specific lineages. This process is regulated by transcription factors, whose functions are confirmed through genetic analyses. The identification of highly conserved, embryonic signaling pathways and transcription regulatory genes illustrates the enormous utility of analyzing embryonic hematopoiesis in frog, chick, fish, and mouse systems to further our understanding of human stem cells.

Animals↗
PubMed1997

Differential regulation of neurogenesis by the two Xenopus GATA-1 genes.

Previously, we have shown that the ventralizing factor bone morphogenetic protein 4 (BMP-4) can inhibit Xenopus neurogenesis. The erythroid transcription factor GATA-1 functions downstream of the BMP-4 signaling pathway and mediates BMP-4-induced erythropoiesis. We have found that similar to BMP-4, GATA-1b inhibits neuralization of Xenopus animal cap (AC) cells. The neural inhibition is not seen with GATA-1a, although both GATA-1a and GATA-1b RNAs are translated at the same efficiency and induce globin expression equally in AC cells. GATA-1b RNA injection into AC cells neither induces expression of Xbra (a general mesoderm marker) nor affects expression of XK81 (epidermal keratin) or BMP-4 and Xvent-1 (two ventral markers). These data suggest that GATA-1b retains the epidermal fate of the AC. Intact GATA-1b protein is required for both inhibition of neurogenesis and induction of globin expression. Our findings indicate that GATA-1b can function in ectoderm to specifically regulate neural inducing mechanisms, apparently related to the expression of chordin, a neuralizing gene. Furthermore, tadpole stage embryos injected with GATA-1b are devoid of all dorsoanterior structures including neural tissue. This report provides evidence that the two transcription factors, derived from a recent genome duplication, share a common biological activity (stimulation of erythropoiesis) while also exhibiting a distinct function (inhibition of neurogenesis).

Animals↗
PubMed1996

The human GATA-6 gene: structure, chromosomal location, and regulation of expression by tissue-specific and mitogen-responsive signals.

GATA factors constitute a family of transcriptional regulatory proteins expressed with distinct developmental and tissue-specific profiles and thought to regulate cell-restricted programs of gene expression. Here we describe the molecular cloning, chromosomal location, and transcription of the human GATA-6 gene. The GATA-6 cDNA encodes a predicted 449-amino-acid protein, which is highly conserved among vertebrates, and includes the two adjacent zinc-finger/basic domains characteristic of the GATA factor family. GATA-6 maps to human chromosome 18q11.1-q11.2 by fluorescence in situ hybridization. The gene is transcribed in a pattern overlapping that of GATA-4. Transcripts for both of these genes are prominent in heart, pancreas, and ovary, but only GATA-6 mRNA is found in lung and liver. GATA-6 transcripts are also detected in cultures of human and rat vascular smooth muscle cells (VSMCs). In VSMCs, GATA-6 transcripts are down- regulated when quiescent cultures are stimulated to proliferate in response to mitogen activation. These data demonstrate that GATA-6 is subject to both tissue-specific and mitogen-responsive regulatory signals. GATA-6 is a prime candidate for a gene that might regulate the differentiative state of VSMCs.

Amino Acid Sequence↗
PubMed1996

Mammalian Cdc42 is a brefeldin A-sensitive component of the Golgi apparatus.

In this study, we have used immunocytochemical and fractionation approaches to provide a description of the localization of the mammalian Cdc42 protein (designated Cdc42Hs) in vivo. A specific anti-peptide antibody was generated against the C-terminal region of Cdc42Hs. Using affinity-purified preparations of this antibody in indirect immunofluorescence experiments, Cdc42Hs was found to be localized to the Golgi apparatus. Similar to the well-characterized non-clathrin coat proteins ADP-ribosylation factor (ARF) and beta-COP, the perinuclear clustering of Cdc42Hs is rapidly dispersed upon exposure of the cells to the drug brefeldin A, suggesting that it too may play a role in the processes of intracellular lipid and protein transport. Employing cell lines possessing inducible forms of ARF, we demonstrate here a tight coupling of the nucleotide-bound state of ARF and the subcellular localization of Cdc42Hs. Specifically, the expression of wild-type ARF had no effect on the brefeldin A sensitivity of Cdc42Hs while, as is the case for ARF and beta-COP, expression of a GTPase-deficient form of ARF (ARF(Q71L)) renders these Golgi-localized proteins resistant to brefeldin A treatment (; ). Moreover, the induced expression of a mutant form of ARF with a low affinity for nucleotide resulted in constitutive redistribution of Cdc42Hs in the absence of brefeldin A treatment. These results suggest that Cdc42Hs may play a role in cell morphogenesis by acting on targets in the Golgi that direct polarized growth at the plasma membrane.

Animals↗
PubMed1996

The Xenopus GATA-4/5/6 genes are associated with cardiac specification and can regulate cardiac-specific transcription during embryogenesis.

The GATA family of nuclear factors has been implicated in the regulation of cell type-specific transcription. We report the isolation of the Xenopus GATA-4 and GATA-6 cDNA clones and characterize the expression patterns of the xGATA-4/5/6 genes. By comparing the sequence of the cDNAs with those previously reported from chick and mammalian sources, we conclude that each is conserved across vertebrate evolution as a distinct gene product. Each gene is expressed in differentiated adult heart and gut, but maintains distinct transcript patterns in various other adult tissues. During embryogenesis, each gene displays a similar overlapping distribution of transcripts localized throughout the developing cardiogenic region. The xGATA-4 gene can be detected in dorsal cardiac progenitor rudiments prior to migration. Axis disruption experiments were used to demonstrate that transcription of these genes is intimately associated with the specification of cardiac progenitors. Ectopic expression of each gene is specifically capable of activating during embryogenesis the transcription of the cardiac genes encoding actin and myosin heavy chain alpha. The data are consistent with a primary role for the GATA-4/5/6 genes in regulating heart development.

Amino Acid Sequence↗
PubMed1996

BMP-like signals are required after the midblastula transition for blood cell development.

We have investigated the process by which the primitive erythroid cells develop during early vertebrate embryogenesis. Cultured Xenopus animal cap (AC) cells transiently activate the transcription of blood cell regulatory genes GATA-1 and GATA-2 but fail to commit stably to the blood lineage. By contrast, cells of the presumptive ventral marginal zone (VMZ), are committed by the midblastula transition (MBT) to express fully on erythroid program. Growth factor BMP-4, a member of the TGF-beta family of signaling molecules, has been implicated in the process of ventral mesoderm patterning. We show that expression of BMP-4 after MBT is sufficient to induce the blood program fully in AC cells. This includes high level expression of the blood markers SCL and globin, which are not activated in AC cells from uninjected embryos. Likewise, expression of a dominant negative receptor after MBT results in relatively normal embryos, which, however, completely lack differentiated blood cells. Our results are consistent with a role for BMP or BMP-like signaling during gastrulation in the differentiation of embryonic blood.

Animals↗
PubMed1996

SSI1 encodes a novel Hsp70 of the Saccharomyces cerevisiae endoplasmic reticulum.

The endoplasmic reticulum (ER) of the budding yeast Saccharomyces cerevisiae contains a well-characterized, essential member of the Hsp70 family of molecular chaperones, Kar2p. Kar2p has been shown to be involved in the translocation of proteins into the ER as well as the proper folding of proteins in that compartment. We report the characterization of a novel Hsp70 of the ER, Ssi1p. Ssi1p, which shares 24% of the amino acids of Kar2p, is not essential for growth under normal conditions. However, deletion of SSI1 results in cold sensitivity as well as enhanced resistance to manganese. The localization of Ssi1p to the ER, suggested by the presence of a conserved S. cerevisiae ER retention signal at its C terminus, was confirmed by subcellular fractionation, protease protection assays, and immunofluorescence. The SSI1 promoter contains an element with similarity to the unfolded protein response element of KAR2. Like KAR2, SSI1 is induced both in the presence of tunicamycin and in a kar2-159 mutant strain, conditions which lead to an accumulation of unfolded proteins in the ER. Unlike KAR2, however, SSI1 is not induced by heat shock. Deletion of SSI1 shows a complex pattern of genetic interactions with various conditional alleles of KAR2, ranging from synthetic lethality to synthetic rescue. Interestingly, SSI1 deletion strains show a partial block in translocation of multiple proteins into the ER, suggesting that Ssi1p plays a direct role in the translocation process.

Amino Acid Sequence↗
PubMed1995

DHEAS as an effective vaccine adjuvant in elderly humans. Proof-of-principle studies.

We have demonstrated that in aged mice, the titer of serum antibody induced against tetanus toxoid correlates with resistance to local paralysis caused by injection of tetanus toxin. Only mice immunized shortly after oral dosing with DHEAS demonstrated high serum antibody titers and complete protection from paralysis. These results became the basis for initiating proof-of-principle studies in human volunteers above age 65 using a licensed influenza vaccine and tetanus toxoid in two independent studies. The use of an oral delivery form of DHEAS before influenza vaccination was associated with a demonstrable increase in the number of individuals with a fourfold increase in HAI titers following vaccination. The overall mean increase in HAI titers was highest in the DHEAS-treated group. The use of DHEAS in the immunization of elderly subjects against tetanus toxoid, while unable to enhance the responses, was not a detriment to antibody response. We conclude that further studies will justify the use of DHEAS as an adjuvant for antigens that represent primary responses in the elderly.

Adjuvants, Immunologic↗
PubMed1995

PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase.

BACKGROUND: Phosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined. RESULTS: We show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate. CONCLUSIONS: These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.

Cell Line↗
PubMed1995

The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos.

Mutations in the human SRY-related gene, SOX9, located on chromosome 17, have recently been associated with the sex reversal and skeletal dysmorphology syndrome, campomelic dysplasia. In order to clarify the role of this gene in skeletal development, we have studied the expression of mouse Sox9 during embryogenesis. Sox9 is expressed predominantly in mesenchymal condensations throughout the embryo before and during the deposition of cartilage, consistent with a primary role in skeletal formation. Interspecific backcross mapping has localized mouse Sox9 to distal chromosome 11. The expression pattern and chromosomal location of Sox9 suggest that it may be the gene defective in the mouse skeletal mutant Tail-short, a potential animal model for campomelic dysplasia.

Amino Acid Sequence↗
PubMed1995

Homotypic interactions of chicken GATA-1 can mediate transcriptional activation.

We used a one-hybrid system to replace precisely the finger II chicken GATA-1 DNA-binding domain with the binding domain of bacterial repressor protein LexA. The LexA DNA-binding domain lacks amino acids that function for transcriptional activation, nuclear localization, or protein dimerization. This allowed us to analyze activities of GATA-1 sequences distinct from DNA binding. We found that strong transcriptional activating sequences that function independently of finger II are present in GATA-1. Sequences including finger I contain an independent nuclear localizing function. Our data are consistent with cooperative binding of two LexA-GATA-1 hybrid proteins on a palindromic operator. The sensitivity of our transcription assay provides the first evidence that GATA-1 can make homotypic interactions in vivo. The ability of a non-DNA-binding form of GATA-1 to activate gene expression by targeting to a bound GATA-1 derivative further supports the notion that GATA-1-GATA-1 interactions may have functional consequences. A coimmunoprecipitation assay was used to demonstrate that GATA-1 multimeric complexes form in solution by protein-protein interaction. The novel ability of GATA-1 to interact homotypically may be important for the formation of higher-order structures among distant regulatory elements that share binding sites for this transcription factor. We also used the system to test the ability of GATA-1 to interact heterotypically with other activators.

Amino Acid Sequence↗
PubMed1995

Effect of acidosis on hydrogen peroxide injury to the isolated perfused rat heart.

We observed that both low and high doses of H2O2 (100 microM and 1 mM, respectively) caused significant and irreversible injury to cardiac contractile function in the isolated perfused heart model. Using 31P-nuclear magnetic resonance spectroscopy, we observed marked metabolic changes following exposure to H2O2, especially at the 1 mM dose. Most remarkable were the increases in the intensity of the phosphomonoester resonance that occurred immediately after exposure to H2O2. The major phosphomonoester species accumulating in hearts exposed to 1 mM H2O2 appears to be AMP. Exposure of hearts to H2O2 in the setting of metabolic acidosis did not significantly alter the functional response of isolated hearts to H2O2. However, the increases in phosphomonoester peak intensity following both doses of H2O2 and the decreases in tissue ATP and total phosphates following 1 mM H2O2 were attenuated by metabolic acidosis.

Acidosis↗
PubMed1995

Negative regulation of chicken GATA-1 promoter activity mediated by a hormone response element.

GATA-1 is a DNA-binding protein that regulates transcription of erythroid-specific genes and is required for the formation of mature erythroid cells. We show here that the GATA-1 hormone response-like element (GHRE) within the first intron of the gene functions as an inhibitory element in chicken erythroid precursor cells, as revealed by expression studies with mutants of the minimal GATA-1 promoter. We identify in these precursor cells the relevant proteins that interact with GHRE as a heterodimer of the thyroid hormone receptor alpha and the chicken ovalbumin upstream promoter transcription factor. Our results indicate that this novel complex can negatively regulate the GATA-1 promoter and suggest that GATA-1 can overcome this inhibitory action. We provide evidence that the viral gene product, v-erb A, can also reduce GATA-1 promoter activity through the GHRE site.

Animals↗