Biomedical subjects

M E Martin

Publications and source records attributed to M E Martin.

At least 37 records · Page 2Linked to original sources

PubMed1999

Corepressor required for adenovirus E1B 55,000-molecular-weight protein repression of basal transcription.

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.

Adenovirus E1B Proteins↗

PubMed1998

Identification of redox-sensitive cysteines in GA-binding protein-alpha that regulate DNA binding and heterodimerization.

The transcription factor GA-binding protein (GABP) is composed of two subunits, GABPalpha and GABPbeta. The DNA-binding subunit, GABPalpha, is a member of the Ets family of transcription factors, characterized by the conserved Ets-domain that mediates DNA binding and associates with GABPbeta, which lacks a discernible DNA binding domain, through ankyrin repeats in the NH2 terminus of GABPbeta. We previously demonstrated that GABP is subject to redox regulation in vitro and in vivo through four COOH-terminal cysteines in GABPalpha. To determine the roles of individual cysteines in GABP redox regulation, we generated a series of serine substitution mutants by site-directed mutagenesis and identified three redox-sensitive cysteine residues in GABPalpha (Cys388, Cys401, and Cys421). Sulfhydryl modification of Cys388 and Cys401 inhibits DNA binding by GABPalpha, whereas, modification of Cys421 has no effect on GABPalpha DNA binding but inhibits dimerization with GABPbeta. The positions of Cys388 and Cys401 within the known Ets-domain structure suggest two very different mechanisms for redox regulation of DNA binding. Sulfhydryl modification of Cys388 could directly interfere with DNA binding or might alter the positioning of the DNA-binding helix 3. Modification of Cys401 may inhibit DNA binding through stabilization of an inhibitory helix similar to that described in the Ets-1 protein. Thus, GABP is regulated through at least two redox-sensitive activities, DNA binding and heterodimerization.

Alkylation↗

PubMed1998

A point mutation in the bulge of the iron-responsive element of the L ferritin gene in two families with the hereditary hyperferritinemia-cataract syndrome.

The molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome is the presence of a mutation in the iron-responsive element (IRE) of the L ferritin gene, located on chromosome 19q13.3-13.4. Two mutations have been reported so far, altering adjacent nucleotides in the IRE loop, in a region that has been extensively studied in vitro and shown to mediate high affinity interaction with the iron-responsive protein. In this report, we describe two families with a new mutation in the bulge of the IRE stem, and we show that this mutation alters the protein-binding affinity of the IRE in vitro to the same extent as the loop mutation. In addition, we present evidence that some variability in the age of onset of cataract can be associated with this genetic syndrome, probably because of additional genetic or environmental factors that modulate the penetrance of the L ferritin defect in the lens. We confirm that the patients do not have increased iron stores despite the persistence of elevated serum ferritin levels and that, accordingly, they do not tolerate well venesection therapy. Further studies will be necessary to elucidate the mechanism responsible for the onset of cataract.

Adult↗

PubMed1998

Novel large apolipoprotein E-containing lipoproteins of density 1.006-1.060 g/ml in human cerebrospinal fluid.

Although the critical role of apolipoprotein E (apoE) allelic variation in Alzheimer's disease and in the outcome of CNS injury is now recognized, the functions of apoE in the CNS remain obscure, particularly with regard to lipid metabolism. We used density gradient ultracentrifugation to identify apoE-containing lipoproteins in human CSF. CSF apoE lipoproteins, previously identified only in the 1.063-1.21 g/ml density range, were also demonstrated in the 1.006-1.060 g/ml density range. Plasma lipoproteins in this density range include low-density lipoprotein and high-density lipoprotein (HDL) subfraction 1 (HDL1). The novel CSF apoE lipoproteins are designated HDL1. No immunoreactive apolipoprotein A-I (apo A-I) or B could be identified in the CSF HDL1 fractions. Large lipoproteins 18.3 +/- 6.6 nm in diameter (mean +/- SD) in the HDL1 density range were demonstrated by electron microscopy. Following fast protein liquid chromatography of CSF at physiologic ionic strength, apoE was demonstrated in particles of average size greater than particles containing apoA-I. The largest lipoproteins separated by this technique contained apoE without apoA-I. Thus, the presence of large apoE-containing lipoproteins was confirmed without ultracentrifugation. Interconversion between the more abundant smaller apoE-HDL subfractions 2 and 3 and the novel larger apoE-HDL1 is postulated to mediate a role in cholesterol redistribution in brain.

Apolipoprotein A-I↗

PubMed1998

Adenovirus E1B 55K represses p53 activation in vitro.

Adenovirus E1B 55K protein cooperates with E1A gene products to induce cell transformation. E1B 55K mediates its effects by binding to and inhibiting the transcriptional activation and growth-suppression functions of the tumor suppressor p53. Previous studies in vivo have suggested that E1B 55K has an active role in repressing p53 transcriptional activation and that this repression function is directed to specific promoters through E1B 55K's interaction with DNA-bound p53. Flag-tagged E1B 55K (e55K) was expressed with the baculovirus expression system and immunopurified. Gel filtration, velocity sedimentation centrifugation, and glutaraldehyde cross-linking indicated that e55K is a dimer with a nonglobular conformation. e55K bound directly to purified p53, causing an approximately 10-fold increase in p53 affinity for tandem binding sites. Using in vitro transcription assays reconstituted with purified p53, e55K, and HeLa cell nuclear extracts, we found that e55K specifically repressed p53 activation. These results demonstrate that as postulated from earlier transient expression experiments, E1B 55K is a specific repressor of transcription from a promoter with bound p53. Since HeLa nuclear extracts contain little detectable histone protein, E1B 55K probably represses transcription through direct or indirect interactions with the RNA polymerase II transcription machinery.

Adenovirus E1B Proteins↗

PubMed1997

GA-binding protein-dependent transcription initiator elements. Effect of helical spacing between polyomavirus enhancer a factor 3(PEA3)/Ets-binding sites on initiator activity.

Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner. The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element. Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns. In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABPalpha and GABPbeta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements. Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Sp1-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.

3T3 Cells↗

PubMed1996

Redox regulation of GA-binding protein-alpha DNA binding activity.

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

3T3 Cells↗

PubMed1996

Induction of Fanconi anemia cellular phenotype in human 293 cells by overexpression of a mutant FAC allele.

The polypeptide encoded by the Fanconi anemia (FA) complementation group C gene, FAC, binds to a group of cytoplasmic proteins in vitro and may form a multimeric complex. A known mutant allele of FAC resulting from the substitution of Pro for Leu at codon 554 fails to correct the sensitivity of FA group C cells to mitomycin C. We reasoned that overexpression of the mutant protein in a wild-type cellular background might induce the FA phenotype by competing with endogenous FAC for binding to the accessory proteins. After stable transfection of 293 cells with wild-type and a mutant FAC allele containing the L554P substitution, four independent clones that expressed four-to-fifteen fold higher levels of transcript from the mutant transgene relative to the endogenous FAC gene showed hypersensitivity to mitomycin C. By contrast, both parental and FAC-overexpressing cells maintained their relative resistance to mitomycin C. No differences in the biosynthesis, subcellular localization and protein interactions of the normal and mutant proteins were detected. The induction of the FA phenotype in this system is compatible with the competition hypothesis and provides support for a functional role of the FAC-binding proteins in vivo.

Alleles↗

PubMed1996

Interactions between phytoestrogens and human sex steroid binding protein.

The interactions of human Sex steroid binding protein (SBP) and the lignans [Nordihydrogaiaretic acid (NDGA) enterolactone (Ent), enterodiol (End)] and isoflavonoid phytoestrogens [Equol (Eq), diazein Dad), genistein (Gen)] were studied. The phytoestrogens had different dose-dependent inhibitory effects on steroid binding by SBP. Their relative efficiencies were: Ent> or = NDGA = Eq > Gen for displacing E2 and Eq > Ent > NDGA > Gen for displacing T. End and Dad were much less active. Scatchard analysis suggested that NDGA had similar non- competitive effects on T and E2 binding by reducing the number of binding sites without changing the association constants. But Eq seemed to inhibit E2 binding non-competitively and T binding competitively. NDGA binding to SBP reduced the immunorecognition of SBP by monospecific anti-SBP antibodies, suggesting that NDGA changed SBP immunoreactivity. Unlike NDGA, Eq binding to SBP caused no immunological changes in SBP, indicating qualitative differences in the effects of the lignan and isoflavonoid. Our results indicate that phytoestrogens may modulate the SBP activity and so influence the role of this protein in the delivery of hormonal information to sex steroid-dependent cells.

4-Butyrolactone↗

PubMed1996

Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH.

At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions.

Adult↗

PubMed1996

Effect of diabetes on protein synthesis rate and eukaryotic initiation factor activities in the liver of virgin and pregnant rats.

To study the effect of prolonged diabetes on protein synthesis and on the activities of initiation factors eIF-2 and eIF-2B in the liver, female rats were treated with streptozotocin. Some animals were mated and studied on day 20 of pregnancy, whereas others were kept virgin and studied in parallel. The protein synthesis rate was measured with an "in vitro' cellfree system, and was lower in diabetic pregnant and virgin animals than in pregnant and virgin controls (30-60%). The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they always showed a higher protein synthesis rate than their mothers or virgin rats. Protein synthesis rate, RNA concentration, and initiation factor 2 activity were higher in pregnant than in virgin rats. Both activity and level of eIF-2 factor changed in parallel to the protein synthesis rate, although no differences could be detected between control and diabetic animals. The eIF-2B activity in tissue extracts from diabetic virgin rats and fetuses was lower than in extracts from their controls, whereas no differences could be detected between pregnant and virgin control rats nor between pregnant control and pregnant diabetic animals. The percentage of the phosphorylated form of eIF-2 factor, eIF-2(alpha P), was slightly lower in virgin than in pregnant rats but was unaffected by the diabetic condition, while in diabetic fetuses this parameter was lower than in their corresponding controls. The cyclic adenosine monophosphate dependent protein kinase level was lower in diabetic rats than in controls, whereas no changes in the activity of casein kinase II were found. The isoelectric forms of the beta subunit of eIF-2 factor, eIF-2 beta, were different in the diabetic and the control animals, indicating that insulin deficiency modifies the phosphorylation of specific substrates. Since no differences were detected in RNA or eIF-2 content between control and diabetic rats, translation may, at least partly, be inhibited in the liver by an impairment of peptide chain initiation caused by the decreased eIF-2B activity which nevertheless is independent of eIF-2 alpha phosphorylation.

Animals↗

PubMed1996

Xanthogranulomatous pyelonephritis in children: conservative management.

OBJECTIVES: Three new cases of xanthogranulomatous pyelonephritis are described in children. METHODS: Laboratory examinations including white and red blood cell counts, erythrocyte sedimentation rate, renal function, urinalysis and cystography were performed. RESULTS: All of the patients had a palpable abdominal mass. Urine culture was positive (Proteus mirabilis) in only 1 patient. Associated urological abnormalities were found in 2 cases. Urographically a functionless kidney was encountered in 2 children and a renal mass in the other. None could be diagnosed preoperatively. CONCLUSIONS: Although nephrectomy has been said to be the treatment of choice, conservative treatment can be successful as shown by 1 of our patients.

Biopsy↗

PubMed1996

E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures.

Lipid peroxidation increases with age in brain and is elevated further in Alzheimer's disease. E-4-hydroxy-2-nonenal and malondialdehyde are products of lipid peroxidation that can adduct and cross-link protein. Neurofibrillary tangles, a feature of Alzheimer's disease composed mostly of tau protein, contain cross-linked and ubiquitin-conjugated protein. In P19 neuroglial cultures, E-4-hydroxy-2-nonenal was a potent cytotoxin that cross-linked cytoskeletal proteins, including tau into high molecular weight species that were conjugated with ubiquitin. Malondialdehyde formed monoadducts with cell protein but did not cross-link and was not cytotoxic. A non-crosslinking analogue of E-4-hydroxy-2-nonenal was not cytotoxic. E-4-Hydroxy-2-nonenal may contribute to neurodegeneration and neurofibrillary tangle formation in Alzheimer's disease.

3,4-Methylenedioxyamphetamine↗

PubMed1995

The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution.

The complete DNA sequence was determined for strain U1102 of human herpesvirus-6, a CD4+ T-lymphotropic virus with disease associations in immunodeficient settings and a possible complicating factor in AIDS. The genome is 159,321 bp in size, has a base composition of 43% G + C, and contains 119 open reading frames. The overall structure is 143 kb bounded by 8 kb of direct repeats, DRL (left) and DRR (right), containing 0.35 kb of terminal and junctional arrays of human telomere-like simple repeats. Since eight open reading frames are duplicated in the repeats, six span repetitive elements and three are spliced, the genome is considered to contain 102 separate genes likely to encode protein. The genes are arranged colinearly with those in the genome of the previously sequenced betaherpesvirus, human cytomegalovirus, and has a distinct arrangement of conserved genes relative to the sequenced gammaherpesviruses, herpesvirus saimiri and Epstein-Barr virus, and the alphaherpesviruses, equine herpesvirus-1, varicella-zoster virus, and herpes simplex virus. Comparisons of predicted amino acid sequences allowed the functions of many human herpesvirus-6 encoded proteins to be assigned and showed the closest relationship in overall number and similarity to human cytomegalovirus products, with approximately 67% homologous proteins as compared to the 21% identified in all herpesviruses. The features of the conserved genes and their relative order suggested a general scheme for divergence among these herpesvirus lineages. In addition to the "core" conserved genes, the genome contains four distinct gene families which may be involved in immune evasion and persistence in immune cells: two have similarity to the "chemokine" chemotactic/proinflammatory family of cytokines, one to their peptide G-protein-coupled receptors, and a fourth to the immunoglobulin superfamily.

AIDS-Related Opportunistic Infections↗

PubMed1995

Effect of streptozotocin diabetes on polysomal aggregation and protein synthesis rate in the liver of pregnant rats and their offspring.

To study the effect of diabetes on hepatic protein synthesis and polysomal aggregation in pregnant rats, female rats were treated with streptozotocin prior to conception. Some animals were mated, and studied at day 20 of pregnancy, whereas, others were studied in parallel under non pregnant conditions. The protein synthesis rate measured with an "in vitro" cell-free system was higher in pregnant than in virgin control rats. It decreased with diabetes in both groups, although values remained higher in diabetic pregnant rats than in the virgin animals. The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they showed a higher protein synthesis rate than either their respective mothers or virgin rats. Liver RNA concentration was higher in control and diabetic, pregnant rats than in virgin rats, and the effect of diabetes decreasing this parameter was only significant for pregnant rats. Liver RNA concentration in fetuses was lower than in their mothers, and did not differ between control and diabetic animals. The decreased protein synthesis found in diabetic animals was accompanied by disaggregation of heavy polysomes into lighter species, indicating an impairment in peptide-chain initiation.

Animals↗

PubMed1995

Postprandial free fatty acids stimulate activity of human corticosteroid binding globulin.

The effect of postprandial variation of free fatty acids (FFA) on serum corticosteroid binding globulin (CBG) properties and cortisol (hydrocortisone) concentrations were explored in 11 women (20-30 yr) during 8 h after an oral load of tallow (26% C16:0, 18% C18:0, and 43% C18:1), oleic-sunflower (oleic-SF; 73% C18:1), sunflower (SF; 67% C18:2), and mixed oil (MO; 39% C18:1 and 48% C18:2). Serum FFA increased little after SF and MO but more than doubled in the late postprandial period (6 and 8 h) after oleic-SF (due to monounsaturated FFA) or tallow (due to saturated and monounsaturated FFA). CBG concentrations remained unchanged, but in relation with the postprandial elevation of serum FFA, CBG binding activity was increased after tallow or oleic-SF as a result of a combined two- to threefold increase in affinity constant and a 50% reduction in binding sites. Immunological and in vitro binding studies showed the changes in CBG behavior to be conformational and to be mediated mainly by monounsaturated FFA, especially C18:1. The modifications of CBG properties were associated with sustained high concentrations of cortisol (suppression of midday decrease) 6 and 8 h after tallow or oleic-SF. Thus dietary FFA may have an impact on bioavailability of glucocorticoids.

Adult↗

PubMed1994

In vivo effect of free fatty acids on the specific binding of glucocorticosteroids to corticosteroid binding globulin and liver receptors in immature rats.

Stimulating lipase activity with heparin (200 IU/kg b.w.) increased the plasma free fatty acid (FFA) concentration of immature rats (15 days). The effect of this elevated FFA concentration on glucocorticoid binding to corticosteroid binding globulin (CBG), and liver cytosol glucocorticoid receptor (GR), was analyzed. The plasma FFA concentration increased 2-fold, 10 minutes (P < 0.001), 20 minutes (P < 0.01), and 60 minutes (P < 0.01) post-heparin. The corticosterone (B) and progesterone concentrations were unchanged 60 minutes post-injection. The binding activity of immature rat CBG for B dropped 50% (P < 0.001) 60 minutes post-heparin injection, decreased B binding and increased plasma FFA were correlated (r = -0.8). The decreased B binding resulted from a 2-fold decrease in the apparent number of CBG binding sites; the affinity constant (Ka) remained unchanged. The liver cytosol endogenous FFA content of immature rats was also increased 2-fold, 60 minutes after heparin-induced lipolysis. The increased cytosol FFA, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor. The decrease resulted from a significantly lower apparent Ka for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between Ka (r = -0.93) and n (r = -0.90) and the increased liver cytosol FFA content. Thus the higher plasma FFA induced in vivo by lipase activation or a standard FFA mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR.

Animals↗

PubMed1994

Nucleotide sequence analysis of a 38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding human cytomegalovirus immediate-early gene homologs and transactivating functions.

Human herpesvirus 6 (HHV-6) is prevalent in the human population, with primary infection occurring early in life. Its predominant CD4+ T-lymphocyte tropism, its ability to activate human immunodeficiency virus type 1 (HIV-1) gene expression in vitro, and its upregulation of CD4 expression has led to speculation that HHV-6 may act as a positive cofactor in the progression of HIV infection to AIDS in individuals infected with both viruses. Previous sequencing studies of restricted regions of the 161.5-kbp genome of HHV-6 have demonstrated unequivocally that it is a member of the betaherpesvirus subgroup and have indicated that the HHV-6 genome is generally collinear with the unique long (UL) component of human cytomegalovirus (HCMV). In the work described in this report we have extended these sequencing studies by determining the primary structure of 38.5-kbp of the HHV-6 genome (genomic position 21.0 to 59.5 kbp). Within the sequenced region lie 31 open reading frames, 20 of which are homologous to positional counterparts in HCMV. Of particular significance is the identification of homologs of the HCMV UL36-38 and US22-type genes, which have been shown to encode transactivating proteins. We show that DNA sequences encoding these HHV-6 homologs were able to transactivate HIV-1 long terminal repeat-directed chloramphenicol acetyltransferase expression in cotransfection assays, thus demonstrating functional as well as structural conservation of these betaherpesvirus-specific gene products. Our data therefore confirm the close relationship between HHV-6 and HCMV and identify putative immediate-early regulatory genes of HHV-6 likely to play key roles in lytic replication and possibly also in the interactions between HHV-6 and HIV in dually infected cells.

Animals↗