Search PubMed⌕ Search

Biomedical subjects

G L Long

Publications and source records attributed to G L Long.

At least 37 records · Page 2Linked to original sources
PubMed1996

Elements within the first 17 amino acids of human osteonectin are responsible for binding to type V collagen.

The region in human osteonectin (ON) responsible for binding to type V collagen has been identified as the first 17 NH2-terminal residues. This conclusion is based upon binding studies with deletion mutants of ON produced in Escherichia coli, in which parts of the first 17 amino acids have been removed. Wild-type ON from E. coli and mammalian cell-derived nonglycosylated ON bind identically to type V collagen and at least twice as effectively as mammalian cell-derived N-glycosylated ON. In previous studies, it was shown that N-glycosylation at residue 99 significantly reduces the capacity of ON to bind to type V collagen. Results reported in this communication demonstrate that the actual binding site on ON for type V collagen is distal from the site of N-glycosylation in terms of amino acid sequence but may be proximal in the folded, fully glycosylated, three-dimensional structure. Consistent with this conclusion is the ability of a synthetic peptide consisting of amino acids 1-17 to specifically inhibit the binding of ON to type V collagen.

Amino Acid Sequence↗
PubMed1996

Identification of two novel point mutations in the human protein S gene associated with familial protein S deficiency and thrombosis.

Individuals with thrombosis who were believed to possess associated familial protein S deficiency were analyzed for mutations in the protein S gene by a two-step process. First, the individuals were analyzed for protein S Pro626 A/G dimorphism in both their genomic DNA and reverse-transcribed (RT) polymerase chain reaction (PCR)-amplified cDNA from peripheral blood cell mRNA. If a heterozygote expressed both alleles at the mRNA level at this site in genomic DNA, a search for point mutations was made by direct cDNA sequencing. RT-PCR amplification of exons 1-6 with mRNA from two twin sisters, each of whom has severe type I protein S deficiency, revealed both larger and smaller fragments in addition to the expected 504-base pair fragment in normal individuals. A donor splicesite mutation at position +4 of the 5' end of intron A was subsequently identified in both sisters and their mother. This mutation would lead to incorrect precursor mRNA splicing and the observed cDNA products. Translation of the altered mRNAs would result in a truncated protein without biological activity. In a second family, cDNA sequencing revealed a T-->G mutation at codon 603 (Ile-->Ser) in exon 15 of the protein S gene in an individual with protein S deficiency (mixed type) and a history of thrombosis. The same mutation was also detected in the proband's mother and grandmother, both of whom also exhibit protein S deficiency and thrombotic disease. This mutation occurs within a disulfide loop of protein S that is believed to be responsible for binding to C4b binding protein and may result in greater affinity between protein S and C4b, consequently leading to thrombotic disease.

Adult↗
PubMed1996

Factor X Stockton: a mild bleeding diathesis associated with an active site mutation in factor X.

A unique blood coagulation factor X variant has been identified in a family with a history of bleeding. Plasma from affected family members had prolonged prothrombin times and activated partial thromboplastin times, low to below normal factor X coagulant activity, and normal factor X antigen levels. Sequencing of DNA from the propositus revealed a single G to A substitution in one allele of factor X at base 964 resulting in an amino acid substitution of Asn for Asp at residue 282. This residue corresponds with the active site Asp102 of chymotrypsin. The substitution eliminates a TaqI restriction site and provided the basis for a screening assay to detect the mutation in polymerase chain reaction (PCR) amplified factor X exon VIII DNA. Fourteen additional family members were identified as having the mutation at base 964. Plasma factor X purified from the proposita using an anti-factor X monoclonal antibody immunoadsorbent exhibited an approximately 50% decrease in specific activity compared with factor X purified from a normal individual in a similar manner. Bleeding in family members with the mutation, termed factor X Stockton, appears to be due to disruption of normal hemostasis by the presence in plasma of circulating abnormal factor X. Factor X Stockton is the first naturally occurring substitution at the active site Asp of a serine protease and underscores the importance of this amino acid residue in factor Xa coagulant activity.

Amino Acid Sequence↗
PubMed1995

Role of N-linked glycosylation in human osteonectin. Effect of carbohydrate removal by N-glycanase and site-directed mutagenesis on structure and binding of type V collagen.

In this study we demonstrate that the binding region of recombinant truncated human bone osteonectin (tHON) for type V collagen resides between amino acids 1 and 146. After removal of oligosaccharide chain structures from tHON, bovine bone osteonectin (BBON) and human platelet osteonectin (HPON) by N-glycanase, their ability to bind to type V collagen is increased, and HPON affinity to collagen V is the same as that of BBON. These data suggest that glycosylation of osteonectin has a direct or regulatory effect on osteonectin binding to collagen V and that the increase in tHON binding upon removal of carbohydrate is the result of a loss of a down-regulation site or direct interference of the carbohydrate at the binding site. To determine the specific role of each N-glycosylation site in tHON, Asn71 and Asn99 were mutated to Gln (N71Q, N99Q) and Thr73 and Thr101 mutated to Ala (T73A, T101A) to selectively inhibit oligosaccharide attachment. The binding affinity of N99Q and T101Q to collagen V is markedly increased over wild-type tHON, whereas N71Q and T73A are the same as wild-type tHON. The doubled mutant (N71,99Q) binds identically to collagen V as N99Q and T101A. These data suggest that only the position 99 glycosylation site (Asn99-X-Thr101) in tHON is important in the reduction of binding of osteonectin to collagen V. Consistent with the binding data is the observation that both the N71Q and T73A mutant proteins migrate on SDS-polyacrylamide gel electrophoresis gels identically to wild-type tHON, suggesting that there is little or no N-glycosylation of residue 71 in wild-type osteonectin.

Amidohydrolases↗
PubMed1995

Structure and organization of the human thrombospondin 3 gene (THBS3).

The promoter/5' flank sequence, cDNA sequence, and exon/intron structures of the human thrombospondin 3 (THBS3) gene have been determined. THBS3 cDNA clones were obtained by PCR amplification of human fetal lung cDNA using THBS3-specific primers. Analysis of cDNA and genomic sequences showed the THBS3 gene to be composed of 23 exons, 1 more than the number of exons in the previously characterized mouse TSP3 gene. The additional exon results from the division of mouse exon F into exons F1 and F2. The cDNA encodes a polypeptide of 956 amino acids that is highly acidic, with a clustering of acidic side chains in the third quarter of the polypeptide. This region corresponds to seven type III (Ca(2+)-binding) repeats, a feature shared with other thrombospondins. In addition to these type III repeats, four type II (EGF-like) repeats and NH2- and COOH-terminal domains are present in thrombospondin 3. The THBS3 and mouse TSP3 genes differ in intron sizes, but exon sequences and sizes and positions of insertion of introns are conserved to a high degree. The structural organization of the THBS3 gene is of interest because of its close proximity to that of metaxin, with which it shares a common promoter sequence, and to the gene encoding glucocerebrosidase, a deficiency in which causes Gaucher disease.

Amino Acid Sequence↗
PubMed1995

Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid and transforming growth factor beta response elements: a novel mechanism of glucocorticoid regulation of eukaryotic genes.

Glucocorticoids have previously have shown to decrease Type I collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type I procollagen gene expression. These latter studies have included uridine incorporation into pro alpha 1 (I) and pro alpha 2 (I) mRNAs and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking region of the pro alpha 1 (I) collagen gene and the reporter gene, chloramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibroblasts that dexamethasone down regulates the promoter activity of the pro alpha 1 (I) collagen gene. The glucocorticoid-mediated down-regulation of procollagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whole ColCat 3.6 plasmid did not eliminate the effect. The possibility existed that another cis-element in the 5' flanking region of the pro alpha 1 (I) collagen gene was also required for the collagen glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate in a decrease of transforming growth factor-beta (TGF-beta) secretion into the media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid receptor binding to the GRE and TGF-beta activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the interaction of these TGF-beta molecules with cellular membrane receptors and subsequent transduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expression through both the GRE and TGF-beta elements. Depression of procollagen gene expression by glucocorticoids through the TGF-beta element is mediated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the basis for a novel mechanism of glucocorticoid-mediator regulation of eukaryotic genes containing the TGF-beta element.

Animals↗
PubMed1995

Biochemical prototype for familial thrombosis. A study combining a functional protein C mutation and factor V Leiden.

Resistance to activated protein C (APC) is associated with a single amino acid substitution in factor V (Arg506-->Gln, factor V Leiden) that results in delayed inactivation of the molecule by APC. The mutation is present in 20% of patients with a first episode of deep venous thrombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein CVermont). In protein CVermont, the substitution Glu20-->Ala alone (rPC gamma 20A) is responsible for the defective anticoagulant properties of PCVermont. It was recently established that a thrombotic episode occurred in 73% of family members who are heterozygous for both a functional protein C gene mutation and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor VR506Q/VaR506Q and rAPC gamma 20A using recombinant APC and natural purified factor VR506Q from patients homozygous for the Arg506-->Gln substitution. While wild-type recombinant APC (rAPC) slowly cleaves and inactivates factor VR506Q and factor VaR506Q, minimal cleavage of membrane-bound factor VR506Q and VaR506Q by rAPC gamma 20A at Arg306 and Arg679 occurs, and no loss in cofactor activity is observed. Our data demonstrate that rAPC gamma 20A cannot inactivate either factor VR506Q or factor VaR506Q at biologically relevant rates because of impaired cleavage at Arg306 and Arg679.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V↗
PubMed1994

Molecular mechanism for familial protein C deficiency and thrombosis in protein CVermont (Glu20-->Ala and Val34-->Met).

The role of two protein C gamma-carboxyglutamic acid domain mutations in familial thrombosis, protein CVermont (Bovill, E. G., Tomczak, J. A., Grant, B., Bhushan, F., Pillemer, E., Rainville, I.R., and Long, G. L. (1992) Blood 79, 1456-1465), was investigated. Two single mutations (Glu20-->Ala and Val34-->Met) and the naturally occurring double mutation were created by site-directed mutagenesis and were expressed in human kidney 293 cells. Purified recombinant protein C with the mutation glutamate to alanine at position 20 is defective in the assays of activated partial thromboplastin time, factor Va inactivation, and fibrinolysis. Mutation from valine to methionine at position 34 has only a minor effect. Activation of Glu20 mutants by thrombin-thrombomodulin was not enhanced by phospholipid vesicles and showed a different calcium dependence compared with the wild type, suggesting that Gla20 is important in the interaction of the protein C Gla domain with a phospholipid-mediated site on the thrombomodulin molecule. Glu20-substituted protein C is not inhibited by calcium ion in its interaction with the calcium-dependent monoclonal antibody H-11, suggesting that this mutation has lost the calcium-induced, lipid-independent conformational transition of the protein C Gla domain. These data indicate that the loss of Gla20 causes the major familial dysfunction of protein C to associate with phospholipid as well as to undergo Ca(2+)-dependent, lipid-independent conformational changes and are consistent with the importance of Gla20 in both external and internal Ca2+ binding based upon the x-ray-derived structure of the homologous Gla domain in bovine prothrombin.

Alanine↗
PubMed1994

Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont.

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma-carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2-hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.

1-Carboxyglutamic Acid↗
PubMed1994

Bleomycin stimulates pro-alpha 1 (I) collagen promoter through transforming growth factor beta response element by intracellular and extracellular signaling.

The role of transforming growth factor beta as a mediator of the fibrogenic effect of bleomycin in lung has been investigated at the transcriptional level. Several constructs containing the rat pro-alpha 1 (I) collagen promoter fused to the chloramphenicol acetyltransferase gene were transfected into rat lung fibroblasts. Both bleomycin and transforming growth factor beta 1 increased promoter activity in fibroblasts transfected with constructs containing the transforming growth factor beta response element. Fibroblasts transfected with a deletion construct that lacks this response element did not respond to either bleomycin or transforming growth factor beta 1. Anti-transforming growth factor beta 1-neutralizing antibodies did not block the increase in promoter activity induced by bleomycin, suggesting intracellular signaling. Mutation of the transforming growth factor beta response element greatly reduced the bleomycin effect, which also infers intracellular signaling. In addition, plasmin added to the media greatly enhanced bleomycin stimulation of promoter activity demonstrating that transforming growth factor beta mediates the bleomycin effect through extracellular signaling.

Animals↗
PubMed1994

Genetic analysis of a large kindred exhibiting type I protein C deficiency and associated thrombosis.

A previously described large Vermont kindred possessing a high incidence of venous thromboembolism with associated Type I protein C deficiency (1) has been genetically analyzed. All nine exons of the protein C gene, including both coding and non-coding regions, have been amplified from blood cell genomic DNA using the Tag DNA polymerase chain reaction (PCR) and primers corresponding to flanking intronic regions, and the products directly sequenced. An initial mutation (C-->T) resulting in Thr298-->Met was observed in one arm of the family exhibiting a history of thrombosis and protein C deficiency and was designated protein CVERMONT IIa. However, examination of the kindred member parent (male) of this arm and members of other arms of the kindred demonstrated that the mutation entered the arm via the genetically unrelated spouse. Further analysis of the father and members of other arms of the kindred revealed a different mutation (C insertion: CAT-->CCAT), resulting in a frameshift beginning at amino acid #107 (His-->Pro) and truncation of the protein at codon #119 of the mature protein. This mutation, called protein CVERMONT IIb, is associated with protein C deficiency and thrombosis throughout the kindred.

Amino Acid Sequence↗
PubMed1994

Structure of mouse protein S as determined by PCR amplification and DNA sequencing of cDNA.

The cDNA sequence of mouse protein S was derived by conventional PCR amplification from liver mRNA, initially using primers derived from the human cDNA sequence, followed by direct DNA sequencing. Seven overlapping PCR fragments covering all of the mature protein, part of the propeptide, and the 3' noncoding region were generated and sequenced. In some cases primers based upon the human cDNA sequence were ineffective. Subsequent successful amplification with mouse-derived primers to the same regions and comparison of the mouse and human sequences in these regions suggest that the failure of the human primers was due to insufficient degree of heterospecies identity. The mouse protein S cDNA sequence of the coding region shares 82% identity to human. The 3' noncoding region of mouse protein S cDNA has several small deletions and insertions compared to human protein S cDNA. Mature mouse protein S consists of 634 amino acids in a single polypeptide chain and displays domain organization similar to that for other species. The amino acid sequence of mouse protein S is about 80% identical to that of other species. Eleven glutamic acid residues were found in the amino terminal region and are predicted to be sites of gamma-carboxylation. Amino acid residues #80-244 are defined as four cysteine-rich repeat sequences homologous to epidermal growth factor. The remainder of the molecule is homologous to plasma sex steroid binding protein. The mouse protein S contains two potential N-glycosylation sites at positions #458 and 468 and is lacking the putative glycosylation site at #490 found in human protein S.

Amino Acid Sequence↗
PubMed1994

[Clinical application of thoracoscopic surgery: a report of 19 cases].

Nineteen cases (14 males and 5 females) had been operated with thoracoscopic surgery. Ten cases of spontaneous pneumothorax and 1 spontaneous hemopneumothorax had been underwent thoracoscopic bullous or bleb resections by Endo-GIA 30 or 60. Four cases of plural effusion including primary and secondary malignancies were performed to be pleurodesis by talc sclerosis. Among 4 cases of lung mass, 3 were operated by wedge resection. Lobectomy was performed by VATS in one case with primary lung cancer (T1N0). VATS were satisfactory by using the 3 small incisions giving the advantage of rapid and complete recovery without any obvious complications. Even no any secondary pneumonia. So far the VATS is not fully accepted in China due to the cost of the disposable instruments.

Adolescent↗
PubMed1994

Homozygous type I protein C deficiency in two unrelated families exhibiting thrombophilia related to Ala136-->Pro or Arg286-->His mutations.

Separate single nucleotide mutations have been identified in two unrelated homozygous type I protein C deficient individuals suffering from thrombophilia. Each mutation, initially established by direct DNA sequencing of polymerase chain reaction amplification products, results in an amino acid substitution. The first mutation (PCClamart) results in an Ala136 to Pro substitution in the protein's second epidermal growth factor-like domain. The second mutation (PCMünchen) results in an Arg286 to His substitution in the serine protease domain. Comparison of the location of these two mutations and the relative conservation of the two regions in homologous vitamin K-dependent plasma proteins is consistent with the difference in severity of protein C deficiency and disease in the two individuals. Both mutations result in the abolition of a naturally occurring restriction endonuclease site, thereby allowing independent confirmation of the mutations and rapid and unambiguous genetic analysis of protein C deficiency in family members. In both families, the genetic analysis has proven useful in cases where an assignment of the protein C status based upon clinical laboratory measurements was either ambiguous or incorrect.

Adolescent↗
PubMed1992

Binding of protein S to C4b-binding protein. Mutagenesis of protein S.

Protein S and C4b-binding protein (C4BP) form a tight complex (Kd approximately 0.6 nM) the physiologic purpose of which is unknown. The participation of protein S in this complex was investigated using site-specific mutagenesis. Normal recombinant human protein S (rHPS) and five specifically mutated protein S analogs were expressed in transformed human kidney 293 cells and the following properties were characterized: solution-phase C4BP binding, ability to be cleaved by thrombin, ability to act as a cofactor in the activated protein C-catalyzed inactivation of factor Va, and gamma-carboxyglutamic acid content. In some cases, beta-hydroxyaspartic acid plus beta-hydroxyasparagine content was also determined. Binding studies indicated that while clearly important for a high affinity interaction, the amino acid sequence Gly605-Ile614 identified by Walker (Walker, F J. (1989) J. Biol. Chem. 264, 17645-17648) does not account for all the binding energy of the HPS-C4BP interaction. All mutants perturbed in this region or lacking it altogether displayed reduced C4BP binding, and some retained anticoagulant cofactor function. Neither human factor X nor human steroid-binding protein had any measurable ability to compete with plasma HPS for C4BP binding. Furthermore, bovine protein S and a rHPS analog with bovine sequence from Gly597-Trp629 bound to human C4BP with the same affinity as did HPS, and both proteins substituted effectively for HPS as a cofactor for activated protein C in an otherwise human anticoagulation system. Together these results suggest that optimal binding of protein S to C4BP requires the putative alpha-helix Gly605-Ile614, as well as other undetermined regions of protein S, and that the regions of HPS responsible for C4BP binding and activated protein C cofactor function are structurally isolated.

1-Carboxyglutamic Acid↗
PubMed1992

Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations.

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.

Adolescent↗