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Biomedical subjects

P F Bray

Publications and source records attributed to P F Bray.

At least 19 recordsLinked to original sources
PubMed1992

Demyelinating disease after neurologically complicated primary Epstein-Barr virus infection.

This report describes five patients who, following a neurologically complicated primary Epstein-Barr virus infection, developed progressive or relapsing neurologic deficits. The sequelae in four patients followed 4 to 12 years led to the diagnosis of multiple sclerosis (MS). The fifth patient presented with acute disseminated sclerosis and exhibits diffuse neurologic deficits that have persisted for 2 years. We suggest that the diagnosis of an unexplained acute neurologic or psychiatric syndrome should raise the question of a primary EBV etiology. A precisely timed serologic and hematologic study of the blood is imperative to capture the essential evidence. The data presented represent a clinical association between a neurologically complicated primary EBV infection and both chronic and acute demyelinating disease. The evidence does not justify a conclusion that EBV virus causes MS.

Adolescent↗
PubMed1992

Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein.

The Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several disparate malignancies. Prior studies on patients with multiple sclerosis (MS) showed that 100% are EBV-seropositive and that their blood contains higher antibody titers than those of controls to both transformation and lytic cycle antigens. We performed three different assays for antibodies in CSF to three major EBV antigens from patients with MS and controls. Among 93 patients with MS, 79 (85%) had CSF that reacted with a 70 kD protein, shown to be the nuclear antigen, EBNA-1, whereas only 11 (13%) of 81 EBV-seropositive controls reacted, p less than 0.001. The CSF of all 14 MS patients, unreactive on immunoblots, contained oligoclonal bands on agarose electrophoresis. Together, the two techniques exhibit 100% sensitivity in the confirmatory diagnosis of MS. We also performed amino acid searches of the Protein Identification Resource sequence database for protein homologies to EBNA. Two pentapeptide identities were found between EBNA-1 and myelin basic protein: QKRPS and PRHRD. None of more than 32,000 other proteins in the database contained both pentapeptides. In healthy EBV-seropositive persons, the EBV-specific, MHC-restricted T lymphocytes keep the EBV-containing B lymphocytes locked in the transformed state. However, in the host genetically susceptible to MS, the same population of lymphocytes might recognize and interact with either of the two identified pentapeptides, inadvertently damaging MBP.

Amino Acid Sequence↗
PubMed1991

Quantitative isolation of RNA from human platelets.

We have adapted the acid-guanidinium-phenol-chloroform extraction procedure of Chomczinsky and Sacchi to achieve efficient rapid recovery of total RNA from human platelets. Sufficient platelet RNA (20 micrograms of total RNA per 30 ml of whole blood) can be recovered from relatively small individual samples to perform Northern blot analysis on individual donors and detect the mRNAs for glycoproteins IIb1(GP IIb) and IIIa1(GP IIIa), 3.4 kb and 6.2 kb, respectively. Platelet GP IIb and GP IIIa mRNAs could also be reverse transcribed, and amplified in vitro by the polymerase chain reaction (PCR). Thus, our technique allows simultaneous Northern blotting and PCR, and therefore should be of great help to the characterization of inherited platelet disorders such as Glanzmann's thrombasthenia.

Blood Platelets↗
PubMed1990

Human platelets and megakaryocytes contain alternately spliced glycoprotein IIb mRNAs.

The earliest marker of the megakaryocyte lineage in human bone marrow is a heterodimeric complex of glycoproteins IIb (GPIIb) and IIIa (GPIIIa). GPIIb-IIIa is a member of the integrin family and serves as a receptor for fibrinogen and other adhesive glycoproteins on the surface of activated blood platelets. Mature platelet GPIIb is processed from a single precursor peptide into a disulfide-linked GPIIb alpha and GPIIb beta chain. To study possible mechanisms which could determine ligand specificity of the GPIIb-IIIa complex we investigated whether there are multiple forms of GPIIb mRNA by screening an unamplified lambda gt10 human erythroleukemia cell cDNA library. Four GPIIb clones were isolated and sequenced. Two contained a 102-nucleotide in-frame deletion corresponding to 34 amino acids in the extracellular domain of GPIIb beta. Polymerase chain reaction amplification of human erythroleukemia cell cDNA confirmed the presence of two different transcripts. Normal genomic DNA sequence was shown to contain consensus splice-donor and splice-acceptor sites indicating a 102-base pair exon that is spliced in or out. Using primers specific for either transcript, both forms of GPIIb mRNA were identified by the polymerase chain reaction in normal human platelets and megakaryocytes, but not in reticulocytes or two white blood cell lines. These data provide evidence for two forms of GPIIb in platelets and megakaryocytes and suggest a mechanism for variations in receptor ligand affinity or localization.

Amino Acid Sequence↗
PubMed1990

Identification of an abnormal gene for the GPIIIa subunit of the platelet fibrinogen receptor resulting in Glanzmann's thrombasthenia.

The platelet fibrinogen receptor, which is composed of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), belongs to a large family of receptors that participate in a multitude of biologically important adhesive interactions. Platelets from most patients with the autosomal recessive bleeding disorder, Glanzmann's thrombasthenia, are deficient in GPIIb and GPIIIa. We have used cDNA probes to analyze the GPIIb and GPIIIa genes in four patients from three kindreds with Glanzmann's thrombasthenia. Southern analysis of their DNA was identical to that observed in normals when probed with a full-length GPIIb cDNA or a 3' GPIIIa cDNA. However, in one family, a 5' 2.0 kb GPIIIa cDNA identified abnormal DNA fragments in the father and two affected siblings' genes. A series of restriction digests resulting in small genomic fragments were probed with portions of the 5' 2.0 kb GPIIIa cDNA and indicated that the abnormal sequences are flanked by normal fragments of the GPIIIa gene. To analyze further the genetic defect in this family, RNA was prepared from their platelets. Northern analysis revealed normal levels of GPIIb mRNA compared to control platelets. We were unable to identify GPIIIa mRNA of any size in the clinically affected family members. We also identified an EcoRI restriction fragment length polymorphism (RFLP) that permitted carrier status determination in the clinically unaffected siblings. These studies indicate that Glanzmann's thrombasthenia can be caused by heterogeneous defects in the GPIIIa gene. Furthermore, we have shown that platelets can be used to characterize normal and abnormal GPIIIa and GPIIb mRNA, and RFLPs may be used to determine the carrier status in some families with Glanzmann's thrombasthenia. The specific gene abnormality in this family appears to represent an example of an insertional mutation resulting in a human disease.

Blood Platelet Disorders↗
PubMed1990

Carotid thrombosis following neck irradiation.

Therapeutic irradiation may accelerate atherosclerosis, increasing the risk of vascular stenosis or occlusion several to many years following radiation. However, intimal damage following irradiation may result earlier in thrombosis without stenosis. This report discusses three cases of carotid occlusion that occurred within 3 years of moderate dose irradiation. Angiographic studies showed that occlusion occurred in the absence of atherosclerotic stenosis. A review of the literature supports the conclusion that people who receive neck irradiation are at risk not only for the delayed development of diffuse atherosclerosis but also for thrombotic occlusion within months to several years. We suggest that patients who develop neurological symptoms or signs following neck irradiation, regardless of age, dose of radiation, or interval since radiation, should be evaluated for carotid or vertebral artery disease.

Adult↗
PubMed1988

Physical linkage of the genes for platelet membrane glycoproteins IIb and IIIa.

The fibrinogen receptor on human platelets is a prototypic member of the integrin family and is composed of subunit glycoproteins IIb (gpIIb) and IIIa (gpIIIa) in a 1:1 stoichiometric ratio. We have isolated cDNA clones for gpIIb and gpIIIa and localized both genes to chromosome 17. In the current study, several approaches were used to localize and map the genes for gpIIb and gpIIIa. A preliminary evaluation of subchromosomal localization was performed by using a panel of mouse-human somatic cell hybrids that contain different amounts of the long arm of human chromosome 17. Southern hybridization to the DNA of these hybrids shows that both genes map near the thymidine kinase gene. In situ hybridization to intact human chromosomes localized both genes to the 17q21-22 region. To better define the physical distance between the two genes, we examined the genomic hybridization pattern of each cDNA probe to high molecular weight restriction fragments separated by pulsed-field gel electrophoresis. Serial hybridizations of the same filter have allowed construction of long-range Mlu I and Sfi I restriction maps spanning more than 500 kilobases. Finally, nonoverlapping portions of the cDNAs for both gpIIb and gpIIIa were used to probe Sfi I digests of genomic DNA separated by field-inversion gels. This confirmed that the genes are physically linked within the same 260-kilobase Sfi I fragment and suggests that the gene for gpIIb is located on the 3' side of the gene for gpIIIa. These results suggest that coordinate expression of gpIIb and gpIIIa may depend on physical proximity.

Animals↗
PubMed1988

Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17.

Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3' untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.

Amino Acid Sequence↗
PubMed1987

Platelet glycoprotein IIb. Chromosomal localization and tissue expression.

The GPIIb-IIIa complex functions as a receptor for cytoadhesive proteins on the platelet surface. Both GPIIb and GPIIIa are synthesized by a human erythroleukemia (HEL) cell line. We isolated several cDNA clones by screening a HEL cell cDNA library with an oligonucleotide derived from amino acid sequence of GPIIb. Nucleotide and amino acid sequences were determined from 703 bp of one of these clones. Amino acid sequence of purified platelet GPIIb peptides confirmed the identity of the clone. The cDNA encodes the carboxyl terminus of the large (alpha) subunit of GPIIb and all of the smaller (beta) subunit of GPIIb. By hybridizing the cDNA directly to chromosomes separated by dual laser chromosome sorting, the gene for GPIIb was mapped to chromosome 17. Northern blot analysis showed a approximately 3.4-kb GPIIb mRNA in HEL cells. We also compared the amino acid sequences determined from eight additional platelet GPIIb peptides with the derived amino acids from a published HEL cell GPIIb cDNA, and the platelet and HEL cell proteins appear to be the same. Despite previous reports that vascular endothelial cells and monocytes contain GPIIb, no GPIIb mRNA was observed in either type of cell. Thus, GPIIb appears to be specific for the platelet-megakaryocyte membrane and is distinct from the alpha subunits of the adhesion receptors in other normal tissues.

Amino Acid Sequence↗
PubMed1986

Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa.

Congenital absence of platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIb and GPIIIa are present as a heterodimeric, noncovalent complex in the platelet plasma membrane and function as the fibrinogen receptor. To characterize synthesis of these two proteins, RNA isolated from a human leukemia cell line that contains GPIIb and GPIIIa was translated in a wheat germ cell-free system. Polyclonal antibodies specific for each protein immunoprecipitated distinct [35S]methionine-labeled precursors, indicating that GPIIb and GPIIIa are translated from separate mRNAs. Moreover, using specific antibodies against either intact unreduced GPIIb or the beta subunit, we obtained evidence for synthesis of a common polypeptide precursor for GPIIb alpha and GPIIb beta. Based on experiments using microsomal membranes, it appears that GPIIb is integrated into the platelet membrane with little or no cytoplasmic component. These results suggest that precursors of GPIIb and GPIIIa may be encoded by separate genes and that each precursor is processed before delivery to the plasma membrane.

Cell Line↗
PubMed1985

Epstein-Barr nuclear antigen and viral capsid antigen antibody titers in multiple sclerosis.

To characterize the antibody response to the Epstein-Barr virus (EBV) in MS, we studied serum anti-EBV nuclear antigen (anti-EBNA) and anti-EBV capsid antigen (anti-EBVCA) titers. Both titers were assayed in 93 age- and sex-matched pairs of MS patients and controls. Anti-EBVCA titers were measured by indirect immunofluorescence and anti-EBNA titers by anticomplement immunofluorescence. The seropositivity rate of both anti-EBVCA and anti-EBNA in MS patients was 100%, compared with 84% in controls (p less than 0.0001). Both anti-EBVCA and anti-EBNA titers were significantly higher in MS patients than in controls (p less than 0.0001). The data suggest that EBV has a significant seroepidemiologic association with MS, but they do not define what role EBV antibodies play in the pathogenesis of the disease.

Adolescent↗
PubMed1979

CT scans in Menkes disease.

The clinical courses and serial computerized tomography (CT) scans of four patients with Menkes disease are described. Although the initial clinical presentations were similar, head growth and serial CT scans showed striking individual differences. The CT scans varied from showing no abnormalities early in the disease to showing diffuse cortical atrophy, subdural accumulation of fluid, or multifocal areas of ischemic infarction. The pathologic findings in one patient showed only cerebral and cerebellar atrophy, whereas the findings in another patient showed areas of ischemic infarction, probably secondary to abnormal vessels. Menkes disease should be suspected in male infants with psychomotor deterioration and seizures, or when trauma is suspected from subdural hematoma and multiple fractures.

Brain↗