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At least 973 records · Page 54Linked to original sources
PubMed2001

Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species.

Vascular endothelial growth factor (VEGF) has been implicated in the reendothelialization of the vascular wall after balloon injury. This study investigated whether thrombin, which is formed during activation of the coagulation cascade at sites of vascular injury, upregulates VEGF expression in vascular smooth muscle cells (VSMCs). VEGF expression was assessed in native and cultured VSMCs by Northern blot analysis and reverse transcription-polymerase chain reaction and the release of VEGF protein by immunoassay. alpha-Thrombin time- and concentration-dependently increased VEGF mRNA levels, mainly that mRNA coding for the soluble splice variant VEGF(164/165), and stimulated the release of VEGF protein. These effects required the proteolytic activity of thrombin and were mimicked by a thrombin receptor activating-peptide. Upregulation of VEGF expression was also induced by conditioned medium from alpha-thrombin-stimulated VSMCs. Both the early and the delayed alpha-thrombin-induced VEGF expressions were attenuated by antioxidants and by diphenyleneiodonium. alpha-Thrombin-induced VEGF release was significantly reduced by a platelet-derived growth factor (PDGF)-, a transforming growth factor (TGF)-beta-, and a basic fibroblast growth factor (bFGF)-neutralizing antibody. Thrombin caused a redox-sensitive upregulation of expression of VEGF in VSMCs through a direct and an indirect effect, which was dependent on the endogenous formation of PDGF, TGF-beta, and bFGF. Upregulation of VEGF expression may represent an important mechanism by which the coagulation cascade contributes to the regeneration of the endothelial lining at sites of balloon injury.

Acetylcysteine↗
PubMed2003

Comparative and functional analysis of cardiovascular-related genes.

The ability to detect putative cis-regulatory elements in cardiovascular-related genes has been accelerated by the availability of genomic sequence data from numerous vertebrate species and the recent development of comparative genomic tools. This improvement is anticipated to lead to a better understanding of the complex regulatory architecture of cardiovascular (CV) genes and how genetic variants in these non-coding regions can potentially play a role in cardiovascular disease. This manuscript reviews a recently established database dedicated to the comparative sequence analysis of 250 human CV genes of known importance, 37 of which currently contain sequence comparison data for organisms beyond those of human, mouse and rat. These data have provided a glimpse into the variety of possible insights from deep vertebrate sequence comparisons and the identification of putative gene regulatory elements.

Animals↗
PubMed2005

High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips.

Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.

Gene Library↗
PubMed1998

The 10Sa RNA gene of Thermus thermophilus.

The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T. thermophilus 10Sa RNA are similar to E. coli 10Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus 10Sa RNA.

Amino Acid Sequence↗
PubMed2026

Epigenetic alterations in rheumatoid arthritis: multilayer mechanisms and translational opportunities.

Rheumatoid arthritis (RA) is a chronic inflammatory disease driven by immune dysregulation, in which genetic susceptibility and environmental exposures promote persistent synovitis, progressive joint damage, and systemic comorbidities. Recent epigenomic studies show several recurring abnormalities. Many RA susceptibility variants lie outside protein-coding sequence and map to immune-cell and synovial fibroblast regulatory elements, linking inherited risk to enhancer activity, methylation quantitative trait effects, and distal gene control. Blood-based epigenome-wide association studies identify disease-associated DNA methylation signatures, but these signals require careful control for leukocyte composition, smoking, treatment exposure, and disease stage. RA fibroblast-like synoviocytes also display stable methylome remodeling, including relative hypomethylation at loci involved in inflammation, migration, matrix degradation, and apoptosis resistance, while TET3-associated 5-hydroxymethylcytosine has emerged as a functional contributor to chemokine production and invasive stromal behavior. Histone modifications, chromatin accessibility, and 3D genome organization define pathogenic regulatory states and connect non-coding risk loci to effector genes in immune and stromal compartments. Finally, miRNAs, lncRNAs, circRNAs, snoRNAs, extracellular RNAs, and m6A-related pathways add post-transcriptional and chromatin-linked layers with potential biomarker value. We synthesize these findings and discuss translational opportunities for diagnosis, stratification, flare monitoring, and therapeutic targeting, while emphasizing incomplete replication, uneven evidence across epigenetic layers, biospecimen variability, and the need for causal, longitudinal, cell-type-resolved validation.

Humans↗
PubMed2004

Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.

A recognition site for the cAMP-dependent protein kinase was introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make a variant of MAb-chCC49 containing a highly stable phosphate. To design this monoclonal antibody (MAb) without changing its immunoreactivity or biological properties, molecular modeling was used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. We selected one position to mutate on the heavy chain based on molecular dynamics study of the solvated antibody. A vector expressing the mutant was constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5. MAb-WW5 contained the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity, and retained the phosphate stably. Compared with MAb-chCC49K1, another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5 showed much improved stability: about a 10-fold increase in resistance to hydrolysis. MAb-WW5 exhibited the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells as we observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.

Adenocarcinoma↗
PubMed2004

Cloning and identification of NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A.

AIM: To clone, identify and study new NS5ATP2 gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A. METHODS: On the basis of subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus, the coding sequence of new gene and its spliced variant were obtained by bioinformatics method. Polymerase chain reaction (PCR) was conducted to amplify NS5ATP2 gene. RESULTS: The coding sequence of a new gene and its spliced variant were cloned and identified successfully. CONCLUSION: A new gene has been recognized as the new target transactivated by HCV NS5A protein. These results brought some new clues for studying the biological functions of new genes and pathogenesis of the viral proteins.

Antigens, Polyomavirus Transforming↗
PubMed2001

The effects of catechol-O-methyltransferase inhibition on estrogen metabolite and oxidative DNA damage levels in estradiol-treated MCF-7 cells.

Many of the major identified risk factors for breast cancer are associated with exposure to endogenous estrogen. In addition to the effects of estrogen as a growth factor, experimental and epidemiological evidence suggest that catechol metabolites of estrogen also contribute to estrogen carcinogenesis by both direct and indirect genotoxic mechanisms. O-Methylation catalyzed by catechol-O-methyltransferase (COMT) is a Phase II metabolic inactivation pathway for catechol estrogens. We and others have found that a polymorphism in the COMT gene, which codes for a low activity variant of the COMT enzyme, is associated with an increased risk of developing breast cancer; therefore, the goal of the current study was to investigate the role of decreased COMT activity on estrogen catechol levels and on oxidative DNA damage, as measured by 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) levels. MCF-7 cells were pretreated with dioxin as a means to increase estrogen metabolism to catechol estrogens, then treated with estradiol (E2) +/- Ro 41-0960, a COMT-specific inhibitor. After extraction from culture medium, estrogen metabolites were separated using an high-performance liquid chromatography-electrochemical detection method. As expected, dioxin dramatically increased E2 oxidative metabolism, primarily to its 2-OH and 2-methoxy metabolites. The COMT inhibitor blocked 2-methoxy E2 formation. This was associated with increased 2-hydroxy E2 (2-OH E2) and 8-oxo-dG levels. In the presence of COMT inhibition, increased oxidative DNA damage was detected in MCF-7 cells exposed to as low as 0.1 microM E2, whereas in the absence of COMT inhibition, no increase in 8-oxo-dG was detected at E2 concentrations < or =10 microM. This study is the first to show that O-methylation of 2-OH E2 by COMT is protective against oxidative DNA damage caused by 2-OH E2, a major oxidative metabolite of E2.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

[Synthesis of hirudin variant 1 (HV1) gene and primary study of expression in yeast].

OBJECTIVE: Hirudin is an extremely efficient and specific thrombin inhibitor. It is clinically used to prevent the formation of thrombus. In this research the hirudin gene was put into yeast system for expression to evaluate the feasibility of artificially synthesized gene expressed in eukaryotic system and study the factor affecting expression level. METHODS: According to the amino acid sequence of hirudin variant 1 (HV1), the genetic code saccharomyces cerevisiae was used to design and synthesize the HV1 gene. Amplified by PCR, it was inserted into cloning vector pBS-SK(+) and sequenced. Ligation with the signal peptide gene of yeast alpha factor the correct HV1 gene was inserted into yeast expression vector pYC-DE. The recombinant plasmid was transformed into the cell of S. cerevisiae BJ1990 to carry out the primary expression experiment. RESULTS: In cultured supernatant of screened positive clone the hirudin activity was detected to be 30 ATU/ml. The expression level was higher than HV2 in yeast and HV1 in prokaryotic system. The N terminal amino acid sequence completely matches with natural hirudin. CONCLUSIONS: It was proved by this study that the synthesized hirudin gene had been expressed in yeast successfully. This result showed that it was a better way to carry out the expression in yeast using synthesized HV1 gene and a stronger promoter.

Base Sequence↗
PubMed1992

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.

Amino Acid Sequence↗
PubMedDate not supplied

Animal models for human craniofacial malformations.

Holoprosencephaly malformations, of which the fetal alcohol syndrome appears to be a mild form, can result from medial anterior neural plate deficiencies as demonstrated in an ethanol treated animal model. These malformations are associated with more medial positioning of the nasal placodes and resulting underdevelopment or absence of the medial nasal prominences (MNPs) and their derivatives. Malformations seen in the human retinoic acid syndrome (RAS) can be produced by administration of the drug 13-cis-retinoic acid in animals. Primary effects on neural crest cells account for most of these RAS malformations. Many of the malformations seen in the RAS are similar to those of hemifacial microsomia, suggesting similar neural crest involvement. Excessive cell death, apparently limited to trigeminal ganglion neuroblasts of placodal origin, follows 13-cis retinoic acid administration at the time of ganglion formation and leads to malformations virtually identical to those of the Treacher Collins syndrome (TCS). Secondary effects on neural crest cells in the area of the ganglion appear to be responsible for the TCS malformations. Malformations of the DiGeorge Syndrome are similar to those of the RAS and can be produced in mice by ethanol administration or by "knocking out" a homeobox gene (box 1.5). Human and animal studies indicate that cleft lips of multifactorial etiology may be generically susceptible because of small MNP)s or other MNP developmental alterations, such as those found in A/J mice, that make prominence contact more difficult. Experimental maternal hypoxia in mice indicates that cigarette smoking may increase the incidence of cleft lip by interfering with morphogenetic movements. Other human cleft lips may result from the action of a single major gene coding for TGF-alpha variants. A study with mouse palatal shelves in culture and other information suggest that a fusion problem may be involved.

Animals↗
PubMed1987

Factor IXAlabama: a point mutation in a clotting protein results in hemophilia B.

Factor IXAlabama is a variant factor IX molecule responsible for a clinically moderate form of hemophilia B. Twenty-five kilobases (kb) of the variant gene, including seven exons coding for the structural protein, were cloned and characterized. The restriction map and the arrangement of coding regions are identical to those of the normal gene. DNA sequence analysis of the coding regions revealed a single base-pair difference between the gene for factor IXAlabama and the normal factor IX gene. An adenine to guanine transition in the first nucleotide of exon d causes the substitution of a glycine codon (GGT) for the normal aspartic acid codon (GAT). This point mutation results in a single amino acid substitution at residue 47 of the zymogen and represents the genetic defect in factor IXAlabama.

Amino Acid Sequence↗
PubMed1982

Preferential paternal X inactivation in extraembryonic tissues of early mouse embryos.

The preferential expression of the maternal X chromosome seen in certain extraembryonic membranes of the mouse was studied by investigating the tissues from which these membranes are derived during early development. The electrophoretic variant of the X-coded enzyme PGK-1 (phosphoglycerate kinase) was used to distinguish the expression of the maternal from the paternal X chromosome in heterozygous females. Both the extraembryonic ectoderm and primary endoderm of 6 1/2-day female egg cylinders gave almost exclusive expression of the maternal form of the enzyme whereas the epiblast gave near equal expression of the two parental alleles. No paternal PGK-1 band could be detected in samples of pooled 3 1/2-day blastocysts, but after 3 or 4 days of culture in vitro a faint paternal band was seen in the resultant outgrowths. The activity of the maternal band in these latter samples had increased greatly from that of the blastocysts, consistent with preferential expression of the maternal Pgk-1 allele in the trophoblastic cells of the outgrowths, while both alleles are expressed in inner-cell-mass cells. The results strongly support the idea that non-random X-chromosome expression is due to preferential paternal X inactivation in trophectoderm (from which extraembryonic ectoderm is derived) and in primary endoderm, and not to cell selection.

Animals↗
PubMed1995

Polymorphisms and probable lack of mutation in the WAF1-CIP1 gene in colorectal cancer.

WAF1/CIP1, a gene up-regulated by p53 encodes an inhibitor of cyclin-dependent kinases. Induction of WAF1/CIP1 in cells with intact p53 is believed to be instrumental in cell cycle arrest and apoptosis caused by DNA damage. In a model system, WAF1/CIP1 has been shown to have tumor suppressive activity. It is not known however whether WAF1/CIP1 is mutated in human primary tumors. Cells from colorectal cancer have been shown to acquire a series of genetic alterations, including frequent p53 mutations. Thus colorectal tumors, particularly those without identified p53 mutations, are good candidate to search for putative WAF1/CIP1 mutations. DNA extracted from 45 tumors, (including 28 tumors for which p53 mutations had previously been searched for and not found) were PCR amplified for exon 2 of WAF1/CIP1. A search for point mutations was performed in each amplified product using a denaturing gradient gel electrophoresis (DGGE) technique which enables the efficient screening of codons 9 to 139 (i.e. 80% of the WAF1/CIP1 coding sequence). Two different DNA variants were identified and shown to be present in constitutional DNAs of the corresponding patients. The first variant, a C to A transversion at codon 31, changes a serine for an arginine and was detected in eight tumors (18% of the cases). The second variant, detected in a single case (2%) is a silent A to T transversion at the third base of codon 91. DNA extracted from 70 unrelated members from the Centre d'Etude du Polymorphisme Humain (CEPH) was screened for these polymorphisms. The ser/arg polymorphism of codon 31 was detected in seven cases (10%) thus suggesting that it is not associated with a marked colorectal cancer predisposition. The polymorphism on codon 91 was not detected. Two additional variants (arginine to histidine at position 67 and threonine to methionine at position 80) were observed once each in the CEPH family members. Somatic mutation of the WAF1/CIP1 gene was not observed, indicating that, unless there are hot spots for mutations outside the screened region, this gene is not a frequent site of point mutation in colorectal cancer.

Base Sequence↗
PubMed1994

Two coding change mutations in the HIS2(2) allele characterize the salivary histatin 3-2 protein variant.

The decoded amino acid sequence of a salivary protein variant, histatin 3-2 (formerly termed Pb c), that is found primarily and in high frequency in Black populations was determined by genomic PCR and direct sequencing of the HIS2(2) allele. Two different mutations that cause coding changes were found in exon 5. The first mutation is a single nucleotide (T-->A) substitution that causes a TAT (Tyr)-->TAA (Stop) change at residue 28. This premature stop mutation results in a 27 amino acid histatin 3-2 protein, which is 5 amino acids smaller than the common histatin 3-1 allelic protein (a product of the HIS2(1) allele). The second mutation, a single nucleotide (G-->A) substitution (located only 19 nucleotides upstream of the first mutation) causes a CGA (Arg)-->CAA (Gln) change at residue 22, which eliminates a proteolytic cleavage site. These two mutations explain the differences in electrophoretic patterns of HIS2(1) versus HIS2(2) coded histatin peptides and may have functional significance. Each mutation alters a different DNA restriction site, and this provides a DNA-based test for the mutations. This test should greatly simplify population and family studies of this protein polymorphism, since the saliva-based test is considerably more problematic. Elucidation here of the derived protein sequence of the variant histatin 3-2 protein may also facilitate functional studies.

Alleles↗
PubMedDate not supplied

Structural and serological analysis of cross-reactive idiotypes: comparison of heavy and light chain families of anti-arsonate antibodies.

The amino acid sequences of VH and VL regions of 16 cross-reactive idiotype-positive (CRI+) and ten CRI-negative (CRI-) monoclonal anti-arsonate antibodies from a range of laboratories were compared. Analysis of the data demonstrates the existence of several pairs of hybridoma proteins (within CRI+ and CRI- families) which have identical or very similar sequences for VH and VL. This strongly suggests the existence of at least 4 germ-line genes in A/J mice coding for CRI+VH, CRI+VL, CRI-VH and CRI-VL regions. The CRI+ family, however, consists of VH structures which represent either (1) germ-line sequences, (2) VHVL variants with a few substitutions, or (3) VHVL variants with extensive substitutions. The latter two consist of antibodies which have lost either the CRI or arsonate binding capacities. The CRI- family consists of two very different groups. The first group appears to be the product of CRI- VHVL germ-line genes and their variants. The second group contains antibodies coded by CRI+ germ-line genes but which, due to somatic diversification, become CRI-. Comparison of VHVL of all anti-arsonate antibodies clearly indicates that light chain selection by the heavy chain is a non-random process. Also, there appears to be a parallel diversification of VH and VL. Comparative data on H and L chain structures and serological evidence obtained by three monoclonal antiidiotypic antibodies indicates that formation of the original cross-reactive idiotypic determinant(s) requires an equal contribution for both H and L chains; moreover, CRI consists of a single (or few) idiotopes.

Amino Acid Sequence↗
PubMed2003

Gene organization of human transporter associated with antigen processing-like (TAPL, ABCB9): analysis of alternative splicing variants and promoter activity.

The gene organization of human TAPL (TAP-like, ABCB9) was determined. The TAPL gene consists of 12 exons including the first non-coding exon on human chromosome 12q23.34. Three alternative splicing variants of the 12th exon have been identified by 3(')RACE using RNA from human cell lines and isolated lymphocytes. As expected from the similarity of the amino acid sequences of TAP1, TAP2, and TAPL, the intron insertion points in these three genes are essentially the same. However, the TAP2 and TAPL genes are closely related, since each has common non-coding exon and splicing isoforms. The novel splicing variants of TAPL termed 12B and 12C have shorter carboxyl terminal amino acid sequences than 12A, reportedly a conserved isoform in rodents and human. The proximal promoter region of the TAPL gene lacks a canonical TATA-box but contains several GC-box elements. The 60bp upstream sequence containing two GC-boxes from the human TAPL transcriptional start site confers basal promoter activity.

ATP-Binding Cassette Transporters↗
PubMed2000

Melanocortin 3 receptor (MC3R) gene variants in extremely obese women.

OBJECTIVE: Following several reports of linkage of obesity related phenotypes to human chromosome 20q we sought to determine whether variations of the melanocortin 3 receptor (MC3R) gene are associated with obesity. DESIGN: We screened the MC3R gene coding region and approximately 2 kb of 5' and 3' flanking sequences for DNA variants in unrelated extremely obese women and average weight controls using polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) analysis and DNA sequencing. SUBJECTS: 124 unrelated extremely obese women (body mass index, (BMI)>/=40 kg/m2) and 85 average weight controls (BMI<27 kg/m2). MEASUREMENTS: Radiation hybrid (RH) mapping was performed to localize the MC3R gene. 5' and 3' flanking sequences of MC3R gene were cloned. PCR-SSCP and DNA sequencing were used to detect mutations in the MC3R gene coding region and flanking sequences. RESULTS: RH mapping localized the MC3R gene to 20q13, between markers D20S100 and D20S149. 1083 bp 5' and 653 bp 3' flanking region of the MC3R gene were cloned. A missense mutation (+241, codon 81 ATT/GTT, Ile-->Val) was found in the MC3R coding region. Four more variants were detected in the 5' flanking sequence: -201(C-->G), -239 (A-->G), -762(A-->T) and -769(T-->C). Compared with controls, no significant allele frequency differences were found. Racial differences were found for the +241, -201, -239 and -762 polymorphisms. CONCLUSIONS: Several sequence variants were found in the MC3R gene coding region and in 5' flanking sequences. However, none of the variants were associated with obesity phenotypes. The linkage of extreme human obesity on 20q13 is likely caused by genes other than MC3R. International Journal of Obesity (2000) 24, 206-210

Adult↗