Biomedical subjects

G I Johnston

Publications and source records attributed to G I Johnston.

At least 19 recordsLinked to original sources

PubMed2004

Time course toxicogenomic profiles in CD-1 mice after nontoxic and nonlethal hepatotoxic paracetamol administration.

Adverse drug reactions are a major clinical problem. Drug-induced hepatotoxicity constitutes a large percentage of these reactions. A thorough understanding of the genetic events, specifically, the early "decision-making" processes underlying biological changes caused by drugs and metabolites, is required. To assist in the understanding of these events, we have employed the model hepatotoxin, paracetamol (APAP), and GeneChip technology to investigate global genetic events seen after nontoxic and toxic doses in the mouse. Mice were dosed [vehicle, nontoxic APAP (1 mmol/kg), and toxic APAP (3.5 mmol/kg)], and individual hepatic RNA samples were hybridized to separate chips to determine interanimal variation. Statistical analysis detected 175 CD-1 mouse genes that were significantly regulated (P < 4.1 x 10(-6)), and nonsignificant genes were discarded. For clarity, the significantly regulated genes were then binned into categories according to their major function-antioxidant, glutathione, metabolism, transcription, immune, and apoptosis. There was no hepatic stress observed after dosing 1 mmol/kg APAP, when measured by serum alanine aminotransferase levels. Hepatic toxicity was observed at both 4 and 24 h after a 3.5 mmol/kg dose of APAP. Time course expression profiles for selected genes have been created. These results demonstrate that most active gene expression occurs around 4 h after a toxic dose of APAP. Down-regulation of these genes is observed over 24 h, coinciding with the development of overt toxicity. These data provide a deeper understanding of the in vivo time course of physiological responses of the liver to chemical stress and provide a logical step forward for the investigation of new chemical entities demonstrated positive in chemically reactive metabolite screens. The complete data set can be viewed at http://www.ebi.ac.uk/arrayexpress/. The accession number is E-MEXP-82.

Acetaminophen↗

PubMed1995

Cloning and pharmacological characterization of human alpha-1 adrenergic receptors: sequence corrections and direct comparison with other species homologues.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in this study compared to previous reports for both human and bovine alpha-1cAR membrane preparations. All six alpha-1AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner, including the cloned human alpha-1a/dAR which had not been expressed previously. In spite of significant sequence differences between human alpha-1ARs and their other species counterparts, previously established ligand selectivity remains fairly comparable. In summary, these data represent the first side-by-side comparison of pharmacological properties between species homologs of alpha-1AR subtypes and should facilitate the development of alpha-1AR subtype selective drugs for clinical use.

Amino Acid Sequence↗

PubMed1991

Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1.

Endothelial-leukocyte adhesion molecule 1 is a cell surface glycoprotein expressed by cytokine-activated endothelium that mediates the adhesion of blood neutrophils. Endothelial-leukocyte adhesion molecule 1 is a member of the selectin family of cell adhesion molecules each of which contain an amino-terminal lectin-like domain, followed by an epidermal growth factor-like domain and a variable number of short consensus repeats similar to those found in complement binding proteins. Genomic clones encoding the ELAM gene were isolated and the organization of the ELAM gene was determined. The gene, which is present in a single copy in the human genome, contains 14 exons spanning about 13 kilobases of DNA. The positions of exon-intron boundaries correlate with the putative functional subdivisions of the protein. Introns are found at similar positions in all of the six complement regulatory repeats, suggesting that these elements arose by internal gene duplication. A consensus TATAA element is located upstream of the transcriptional start site. The ELAM promoter contains an inverted CCAAT box and consensus NF-kappa B- and AP-1-binding sites. The ELAM gene was assigned to the q12 greater than qter region of human chromosome 1 by analysis of human-mouse hybrid cell lines. Two other members of the selectin gene family, the leukocyte adhesion molecule 1 (LAM-1, TQ1, LEC-CAM 1, or Leu-8) and the granule membrane protein 140 (GMP-140, PADGEM, or CD62) have been localized to the long arm of chromosome 1, as have the structurally related complement binding proteins, suggesting that these genes may share a common evolutionary history.

Amino Acid Sequence↗

PubMed1990

Structure of the human gene encoding granule membrane protein-140, a member of the selectin family of adhesion receptors for leukocytes.

GMP-140, an inducible granule membrane protein of platelets and endothelial cells, is a member of the selectin family of cell surface receptors that mediate interactions of leukocytes with the blood vessel wall. These molecules all contain an N-terminal lectin-like domain, followed by an epidermal growth factor-like domain, a variable number of consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a cytoplasmic tail. Two variant cDNAs for GMP-140 have been identified, one predicting a soluble form of the molecule lacking the transmembrane domain and the other predicting a molecule containing eight instead of nine consensus repeats. Here we describe the organization of the human gene encoding GMP-140, which spans over 50 kilobase pairs and contains 17 exons. Almost all exons encode distinct structural domains, including the lectin-like domain, the epidermal growth factor-like domain, each of the nine consensus repeats, and the transmembrane region. Each of the two deletions found in the variant cDNAs is precisely encoded by an exon, suggesting that these forms of GMP-140 are derived from alternative splicing of mRNA. By using the polymerase chain reaction, transcripts encoding the putative soluble form of GMP-140 can be amplified from both platelet and endothelial cell RNA. The structure of the GMP-140 gene supports the concept that the selectins evolved as a result of exon duplication and rearrangement.

Amino Acid Sequence↗

PubMed1990

Genomic organization of the selectin family of leukocyte adhesion molecules on human and mouse chromosome 1.

A structurally and functionally related group of genes, lymph node homing receptor (LHR), granule membrane protein 140 (GMP-140), and endothelial leukocyte adhesion molecule 1 (ELAM-1) are shown to constitute a gene cluster on mouse and human chromosome 1. In situ hybridization mapped GMP-140 to human chromosome 1 bands 21-24 consistent with chromosomal localization of LHR. Gene linkage analysis in the mouse indicated that these genes and serum coagulation factor V (FV) all map to a region of distal mouse chromosome 1 that is syntenic with human chromosome 1, with no crossovers identified between these four genes in 428 meiotic events. Moreover, long range restriction site mapping demonstrated that these genes map to within 300 kb in both the human and mouse genomes. These data suggest that LHR, ELAM-1, and GMP-140 comprise an adhesion protein family, the selectins, that arose by multiple gene duplication events before divergence of mouse and human. Furthermore, the location of these genes on mouse and human chromosome 1 is consistent with a close evolutionary relationship to the complement receptor-related genes, which also are positioned on the same chromosomes in both species and with which these genes share a region of sequence homology. These data characterize the organization of a genomic region that may be critical for intercellular communication within the immune system.

Animals↗

PubMed1989

Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation.

GMP-140 is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, it is rapidly redistributed to the plasma membrane. The cDNA-derived primary structure of GMP-140 predicts a cysteine-rich protein with multiple domains, including a "lectin" region, an "EGF" domain, nine tandem consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Some cDNAs also predict a soluble protein with a deleted transmembrane segment. The domain organization of GMP-140 is similar to that of ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils. This similarity suggests that GMP-140 belongs to a new family of inducible receptors with related structure and function on vascular cells.

Amino Acid Sequence↗

PubMed1989

Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells.

GMP-140 is an integral membrane glycoprotein of apparent Mr = 140,000 located in secretory storage granules of platelets and vascular endothelial cells. When these cells are activated, GMP-140 redistributes from the membrane of the granules to the plasma membrane. To gain insight into the potential function of GMP-140, we examined aspects of its structure and biosynthesis. The amino acid composition of platelet GMP-140 revealed elevated numbers of cystinyl (6.1%), prolinyl (7.2%), and tryptophanyl (2.1%) residues. GMP-140 contained 28.8% carbohydrate by weight, distributed among N-acetylneuraminic acid, neutral sugar, and N-acetylglucosamine residues. Enzymatic removal of N-linked oligosaccarides reduced the protein's apparent Mr by more than 50,000. The biosynthesis of GMP-140 in HEL cells, which share biochemical features with megakaryocytes, was studied by pulse-chase labeling with [35S]cysteine followed by immunoprecipitation. HEL cells synthesized a heterogeneous GMP-140 precursor of 98-125 kDa which converted to a mature 140-kDa form within 40-60 min. Removal of high mannose oligosaccarides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent Mr of the precursor but not the mature protein. Tunicamycin-treated HEL cells synthesized three to four precursors of 80-92 kDa, suggesting the possibility of heterogeneity of GMP-140 at the protein level. Exposure of activated platelets to proteases followed by Western blotting indicated that most of the mass of GMP-140 was located on the extracytoplasmic side of the membrane. Our studies indicate that GMP-140 is a cysteine-rich, heavily glycosylated protein with a large extracytoplasmic domain. These features are compatible with a receptor function for the molecule when it is exposed on the surface of activated platelets and endothelial cells.

Amino Acids↗

PubMed1988

Identity of saturable calcium-binding sites on blood platelets and their involvement in platelet aggregation.

Extracellular Ca2+ ions are required for platelet aggregation and we show that they enter two platelet pools. One pool is rapidly filled and easily displaced by EGTA. The second is filled more slowly and is not displaced by EGTA. The EGTA-displaceable pool is believed to be surface-located and was found to contain at least one class of saturable binding sites as well as a class of non-saturable binding sites. The saturable sites were found to be highly selective for Ca2+ (dissociation constant, 3.5 X 10(-7) M) even in the presence of 1 mM Mg2+ ions, and they took up between 261,000 and 307,000 Ca2+ ions/platelet. Full occupancy of the saturable binding sites appeared to be necessary for platelet aggregation to proceed. We also studied platelets that were unable to aggregate normally, either due to the congenital bleeding disorder Glanzmann's thrombastenia or due to experimental manipulation. In both cases we found decreased Ca2+ uptake specifically by the saturable Ca2+ binding sites, and that this was associated with decreased number of GP IIb/IIIa molecules expressed on these platelets. We suggest that the Ca2+ binding sites involved in platelet aggregation are located on the GP IIb/IIIa complexes and may be involved in holding the glycoproteins in the complex together, and that the binding sites need to be fully occupied before aggregation can proceed.

Binding Sites↗

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↗

PubMed1987

Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry.

Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

Antibodies, Monoclonal↗

PubMed1987

Platelet aggregation in whole blood from patients with Glanzmann's thrombasthenia.

We examined platelet aggregation in platelet-rich plasma (PRP) and in whole blood from two patients with Glanzmann's thrombasthenia. In PRP, aggregation was measured by monitoring the changes in light absorbance that occurred in response to aggregating agents; to measure platelet aggregation in whole blood, we used a platelet counting technique. In PRP, the patients' platelets showed defective aggregation in response to ADP, adrenaline, arachidonic acid (AA), and collagen, but normal agglutination occurred in response to ristocetin. In whole blood, however, platelet aggregation in response to the aggregating agents appeared to be either very similar to that which occurred in blood from normal subjects or only slightly reduced. There was a reduced response to all concentrations of ADP and to low concentrations of collagen but a normal response to all concentrations of adrenaline, AA, and higher concentrations of collagen. Conversely, there seemed to be an increased agglutination response to ristocetin. The abnormality in our two patients with Glanzmann's thrombasthenia probably lies in the inability of their platelets to form large, macroscopic aggregates rather than in platelet aggregation per se.

Adenosine Diphosphate↗

PubMed1985

Effects of diamide and iodoacetamide on the expression of the glycoprotein IIb/IIIa complex on blood platelets.

We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins.

Antibodies, Monoclonal↗

PubMed1984

The expression of glycoproteins on single blood platelets from healthy individuals and from patients with congenital bleeding disorders.

Glycoproteins present on the surface of blood platelets are fundamental to normal blood platelet behaviour. We have used monoclonal antibodies and flow cytofluorimetry to study the expression of glycoproteins on single platelets from normal subjects, and from patients with Glanzmann's thrombasthenia and the Bernard-Soulier syndrome. We show that normal platelets are heterogeneous in that individual cells display markedly different numbers of glycoprotein IIb/IIIa complex and glycoprotein Ib molecules. We also show that the two congenital bleeding disorders are associated with markedly reduced numbers of glycoprotein IIb/IIIa complex or glycoprotein Ib molecules on all the platelets rather than the difference residing in a sub-population.

Blood Platelets↗

PubMed1968