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Biomedical subjects

G J Roth

Publications and source records attributed to G J Roth.

At least 37 records · Page 2Linked to original sources
PubMed1993

Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins.

Human platelet glycoprotein (GP) V (M(r) 83,300), whose primary structure is reported here, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome. With PCR techniques and platelet cDNA templates, 1.4 kb of GP V cDNA sequence was obtained that encodes 469 GP V amino acids. A genomic 3.5-kb BamHI fragment was then isolated that includes 3.46 kb of GP V cDNA sequence: the 1.7-kb open reading frame plus 2 bases of the 5' and 1.8 kb of the 3' untranslated regions. Northern blot analysis reveals three GP V platelet transcripts of 3.8, 4.2, and 5.2 kb. A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a "flank-LRG center-flank" array [Roth, G. J. (1991) Blood 77, 5-19] along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries.

Amino Acid Sequence↗
PubMed1993

Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor.

Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.

Base Sequence↗
PubMed1993

Characterization of the gene encoding human platelet glycoprotein IX.

Glycoprotein IX is a relatively small (M(r) 20,000) surface glycoprotein of human platelets; one of three (Ib alpha, Ib beta, IX) polypeptide chains present in the glycoprotein Ib-IX complex that functions as the von Willebrand factor receptor and mediates platelet adhesion in the arterial circulation. Using a cDNA for human glycoprotein IX as the probe, clones were isolated from a human genomic library, and the genomic sequence for glycoprotein IX (3.2 kilobases) was determined. The transcriptional start site was located by RNase protection and primer extension experiments. The gene includes three exons and two introns within 1.6 kilobases of DNA, and the entire open reading frame for glycoprotein IX is included within the third exon. The genes for glycoproteins IX and Ib alpha share similar exon sequences on the 5' side of their ATG start codons, and both genes possess introns in this region. The glycoprotein IX gene contains two consensus regulatory sequences (GATA and ACTTCCT [ets]) in its promoter region (5' flank, within 67 bases of the start site) that are also present in similar sites in the previously described "megakaryocyte-platelet" genes (glycoprotein IIb, platelet factor 4, beta-thromboglobulin: human and rat). Thus, the glycoprotein IX gene shares structural features with other megakaryocyte-platelet genes and contains at least two consensus cis-acting regulatory elements that may govern gene expression.

Amino Acid Sequence↗
PubMed1992

Platelets and blood vessels: the adhesion event.

Circulating blood platelets attach to damaged blood vessels by specific adhesion molecules. In the human arterial circulation, a unique 'shear-dependent' mechanism governs adhesion by a single receptor-ligand (glycoprotein Ib-von Willebrand factor) pair. Here, Gerald Roth reviews recent data that provide insights into the functions of these molecules.

Amino Acid Sequence↗
PubMed1991

The effects of changes in health care delivery on the reported incidence of cutaneous melanoma in western Washington State.

The reported incidence of malignant melanoma of the skin has steadily increased in the United States during the past three decades. Since the treatment of melanoma increasingly has been provided solely on an outpatient basis in recent years, it is likely that some cases of this disease are being missed by population-based tumor registries that do not consistently ascertain cases from sources outside the hospital setting. The population-based tumor registry of western Washington state has been identifying office-treated cases of cutaneous melanoma from records of private and hospital pathology laboratories. However, since laboratory records have often lacked information on place of residence, these cases have not been previously included in the calculation of incidence rates. In the present study, by determining the place of residence of these cases at the time of their diagnosis (by contacting the physician listed on their pathology reports), the authors found that there had been a growing amount of underreporting of cutaneous melanoma from 1974 through 1984. In western Washington state, the incidence of melanoma of the skin had been underestimated in registry rates by approximately 2% in 1974 and approximately 21% in 1984. This trend in the level of underenumeration has spuriously blunted the true increasing incidence of melanoma and may limit the ability to monitor and study this disease in the future.

Delivery of Health Care↗
PubMed1990

Human platelet glycoprotein IX. Characterization of cDNA and localization of the gene to chromosome 3.

Overlapping cDNAs encoding human platelet glycoprotein (Gp)IX were cloned from a human erythroleukemia cell lambda gt11 library. The possibly 'full-length' cDNA of 896 base pairs (bp) includes an open reading frame (528 bp), both 5' (222 bp) and 3' (146 bp) noncoding regions, and a poly(A) tail. Translation predicts a signal peptide of 16 amino acids and a mature protein of 160 amino acids that includes a 24 amino acid leucine-rich glycoprotein (LRG) segment. Southern blot analysis suggests the presence of a single copy of the Gp IX gene, and hybridization of Gp IX cDNA to sorted human chromosomes localizes the Gp IX gene to chromosome 3.

Amino Acid Sequence↗
PubMed1990

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion.

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.

Amino Acid Sequence↗
PubMed1990

Liver histology in patients receiving low dose pulse methotrexate for the treatment of rheumatoid arthritis.

The liver histology of 52 patients treated with intermittent low dose pulse methotrexate for rheumatoid arthritis was evaluated using a modification of the Roenigk grading system. Patients studied had had an average of 3.2 years of treatment or had received 1.7 g methotrexate. No patient had cirrhosis; 15 (29%) patients had evidence of mild fibrosis. Histological abnormalities were not predicted by liver function test changes, with the exception that hypoalbuminaemia occurred in 60% of those with grade IV (modified criteria) findings. The need for liver biopsy in patients with rheumatoid arthritis treated with methotrexate before two years or 1500 mg of treatment has not been established. Whether serial liver biopsies will be needed beyond this time has yet to be determined.

Adult↗
PubMed1989

Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA.

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.

Blood Platelets↗
PubMed1989

Synergy of interleukin 1 (IL-1) with arachidonic acid and A23187 in stimulating PGE synthesis in human fibroblasts: IL-1 stimulates fibroblast cyclooxygenase.

The stimulation of prostaglandin E (PGE) synthesis by interleukin 1 (IL-1) has important physiologic effects in many target tissues. The mechanism(s) whereby IL-1 stimulates PGE synthesis is not well understood. Two alternative mechanisms have been postulated: hydrolysis of membrane phospholipid with release of arachidonic acid substrate and induction of cyclooxygenase activity. We examined these mechanisms in IL-1 stimulation of human dermal fibroblast PGE synthesis. IL-1 failed to induce release of previously incorporated radiolabeled arachidonic acid from fibroblasts under conditions where there was a 30-fold or greater stimulation of PGE synthesis. The calcium ionophore, A23187, gave much less stimulation of PGE synthesis while inducing 30-100% release of incorporated [14C]arachidonic acid. There was marked synergy between IL-1 and agents which increased availability of arachidonic acid. For example, the combination of IL-1 and A23187, used at concentrations where neither agent alone stimulated PGE synthesis, increased PGE synthesis from 3.1 to 22.5 ng/ml; similar synergy was seen between IL-1 and exogenous arachidonic acid. To examine a possible effect of IL-1 on cyclooxygenase synthesis, fibroblast cyclooxygenase was measured by radioimmunoassay. Following treatment with IL-1, fibroblasts demonstrated a two- to threefold increase in content of immunoreactive cyclooxygenase. These results suggest that IL-1 increases fibroblast PGE synthesis by a mechanism(s) other than making available increased membrane arachidonic acid, and that at least part of its effect may be mediated by induction of cyclooxygenase. Furthermore, the effect of IL-1 on fibroblast PGE synthesis is markedly potentiated by agents which increase available substrate.

Arachidonic Acid↗
PubMed1989

cDNA sequence for the alpha M subunit of the human neutrophil adherence receptor indicates homology to integrin alpha subunits.

The receptor on human neutrophils (polymorphonuclear leukocytes) that mediates cellular adherence consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, Mol alpha, or CD11b; Mr, 170,000) and beta (Mac-1 beta, Mol beta, or CD18; Mr, 100,000). We isolated a cDNA clone for the human neutrophil alpha M subunit by screening a lambda gt 11 cDNA library made from chronic myelogenous leukemia neutrophils by using an affinity-purified rabbit polyclonal antibody directed against the alpha M subunit. We used this cDNA clone to obtain additional clones from cDNA libraries made from differentiated HL-60 promyelocytic leukemia cells. Together these cDNAs constitute the complete 1137-amino acid sequence for the mature human alpha M subunit protein. The deduced amino acid sequence indicates the presence of an extensive extracellular domain with three putative metal-binding regions, (i) an amino acid region that is homologous to the A domain of von Willebrand factor, (ii) a 26-amino acid hydrophobic sequence that is a potential transmembrane domain, and (iii) a 19-amino acid cytoplasmic region. The amino acid sequence for the human neutrophil alpha M subunit contains regions that are closely related to amino acid sequences of adhesion receptors belonging to the integrin family.

Amino Acid Sequence↗
PubMed1989

Human platelet glycoprotein IX: an adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures.

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex, GPIb (Mr, 165,000). Using antibody screening, we cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage lambda gt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acid followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. The predicted amino acid sequence of mature GPIX includes an NH2-terminal extracytoplasmic domain of 134 residues, a transmembrane domain of 20 residues, 6 intracytoplasmic residues, and 1 N-linked glycosylation site. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. "Flanking" sequences of approximately 22 amino acids are present at the NH2 and/or COOH sides of the "central" LRG sequence(s) in GPIX, GPIb, and the other human and Drosophila members of the LRG family. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of human platelet glycoprotein IX.

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.

Amino Acid Sequence↗
PubMed1988

Case-control study of borderline ovarian tumors: reproductive history and exposure to exogenous female hormones.

Borderline ovarian tumors possess many of the same morphological features as their malignant counterparts, but they do not destructively invade the ovarian stroma, and the women in whom they develop generally have a favorable prognosis. Female residents of three urban counties of western Washington diagnosed with serous and mucinous borderline ovarian tumors between 1980 and 1985 (n = 116) were interviewed regarding past reproductive events. A random sample of women from the same counties (n = 158) was identified through random digit dialing and were interviewed. The risk of these ovarian tumors among women who had given birth to 1 or 2 children and to 3 or more children was, respectively, 0.7 and 0.4 that of nulliparous women. There was no consistent influence of increasing age at first live birth. Adjusting for parity, a history of lactation was associated with a 50% reduction in risk. Among nulliparous women, a further increase in risk was present in those who reported a history of infertility. Use of oral contraceptives was associated with a 60% reduction in risk. However, the size of the association was not dependent on duration, age at first use, or years since last usage. In conclusion, borderline tumors appear to have similar epidemiological patterns with regard to reproductive events as their more malignant counterparts.

Adult↗
PubMed1988

The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence.

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of the alpha and beta chains of human platelet glycoprotein Ib.

Human platelet glycoprotein Ib has been purified in milligram amounts from platelets obtained by pheresis of thrombocytotic donors. Purification steps included lectin (wheat germ agglutinin) and immuno (murine monoclonal anti-glycoprotein Ib antibody)-affinity chromatography. The disulfide-linked individual alpha and beta chains of GPIb were separated and the amino-terminal amino acid sequence of each chain was determined. Rabbit polyclonal antibody directed against each individual chain was prepared by affinity chromatography and shown to be monospecific by western blot analysis using whole platelet lysate as antigen. These studies outline a useful approach to isolate and characterize the individual chains of GPIb.

Amino Acid Sequence↗