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Biomedical subjects

B M Martin

Publications and source records attributed to B M Martin.

175 records · Page 10Linked to original sources
PubMed1976

Equilibrium binding of thrombin to platelets.

Binding of human [125I]thrombin to washed human platelets was studied in order to analyze the nonenzymic aspects of the thrombin stimulation of platelets. Highly purified alpha-thrombin that was iodinated with lactoperoxidase retained full clotting and esterase activities and full activity toward platelets, was not distinguished from native thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel chromatography, and bound to platelets the same as unlabeled thrombin. Bound and free [125I]thrombin were measured after rapid separation of platelets from the suspending medium by centrifugation through oil. Maximum binding was within 15 s, the shortest time measured. At concentrations of thrombin sufficient to cause less than maximal platelet stimulation, 90% of the total thrombin was free in the suspending solutions. Equilibrium binding was established, with both free thrombin and free platelets retaining activity, and with rapid reequilibration after dilution or addition of unlabeled thrombin. The equilibrium was complex, with the apparent number of binding sites and dissociation constants dependent on thrombin concentration. Analysis of bound thrombin as a function of thrombin concentration by double-reciprocal and Scatchard plots indicated 300-400 high affinity sites (Kdiss = 1.8-2 nM); these correlate with thrombin stimulation of Ca2+ secretion, which shows half maximal effect at 1.5 nM thrombin and maximal effect with 500-600 thrombins bound per platelet.

Binding Sites↗
PubMed1975

Platelet stimulation by thrombin and other proteases.

The mechanism of stimulation of platelets by thrombin and other proteases was studied by following kinetics of secretion of Ca2+ or ATP. The progress-time curves of secretion were analyzed for rate and total amount released. The reaction of thrombin was perturbed by addition of hydroxylamine or a competitive inhibitor and by variation of pH and it was compared with the reactions of other proteases. Trypsin and papain, with specificities for arginyl residues, induced secretion with a time course that was nearly identical with that induced by thrombin when saturating levels of enzyme were used. At low levels of enzyme, trypsin and papain gave extended lags in the progress-time curves. Higher concentrations of trypsin and papain were required for saturation of the measured parameters. Human plasmin (lysly specificity) and bovine chymotrypsin (aromatic amino acid specificity) failed to induce platelet secretion. Active site inhibited thrombin was also ineffective. Both yield and kinetics depended on pH, with the pH profile for each enzyme similar to its profile for hydrolysis of synthetic substrates. Studies at low pH also showed that the early part of the reaction undergoes a change in rate-determining step from enzyme dependent at low enzyme to enzyme indepdenent at high enzyme. Hydroxylamine, a nucleophile that would be expected to accelerate hydrolytic reactions, actually decreased both the rate of initial reactions and yield. A competitive inhibitor of thrombin also decreased both rate and yield; a calculated inhibition constant was in agreement with the value for a synthetic substrate, suggesting that the interaction of thrombin with platelets is analogous to reaction with substrates. A modification of our previous model is proposed in order to accommodate the results described here and to reaoncile the apparent contradictions that enzyme was found not to turn over in the reaction (Detwiler, T. C., and Feinman, R. D. (1973), Biochemistry 12, 282), that catalytic activity is required (Davey, M. G., and Luscher, E. F. (1967), Nature (London) 216, 875; this paper), and that the reaction is characterized by an apparent equilibrium binding (Tollefsen, D. M., Feagler J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). The essential feature is a reversible catalytic step with no dissociation of enzyme from product. This is followed by irreversible, thrombin-independent platelet processes leading to secretion, with yield dependent on the equilibrium concentration of the thrombin product. The model thus has aspects of catalysis, stoichiometry, and an agonist-receptor equilibrium.

Adenosine Triphosphate↗
PubMedDate not supplied

A biochemical and ultrastructural evaluation of the type 2 Gaucher mouse.

Gaucher mice, created by targeted disruption of the glucocerebrosidase gene, are totally deficient in glucocerebrosidase and have a rapidly deteriorating clinical course analogous to the most severely affected type 2 human patients. An ultrastructural study of tissues from these mice revealed glucocerebroside accumulation in bone marrow, liver, spleen, and brain. This glycolipid had a characteristic elongated tubular structure and was contained in lysosomes, as demonstrated by colocalization with both ingested carbon particles and cathepsin D. In the central nervous system (CNS), glucocerebroside was diffusely stored in microglia cells and in brainstem and spinal cord neurons, but not in neurons of the cerebellum or cerebral cortex. This rostralcaudal pattern of neuronal lipid storage in these Gaucher mice replicates the pattern seen in type 2 human Gaucher patients and clearly demonstrates that glycosphingolipid catabolism and/or accumulation varies within different brain regions. Surprisingly, the cellular pathology of tissue from these Gaucher mice was relatively mild, and suggests that the early and rapid demise of both Gaucher mice and severely affected type 2 human neonates may be the result of both a neurotoxic metabolite, such as glucosylsphingosine, and other factors, such as skin water barrier dysfunction secondary to the absence of glucocerebrosidase activity.

Animals↗
PubMedDate not supplied

The genomic structure and developmental expression patterns of the human OPA-containing gene (HOPA).

We determined the genomic organization of the human OPA-containing gene (HOPA) and characterized its developmental expression. The gene encoding HOPA, which contains a rare polymorphism tightly associated with non-specific mental retardation, is 25 kb in length and consists of 44 exons. A promoter scan analysis demonstrates two possible transcription initiation sites without TATA boxes upstream from the putative translation initiation start site. Several informative polymorphisms are evident in the sequence including a large pentanucleotide repeat. Northern blot analysis of the gene transcript and its murine orthologue, MOPA-1, demonstrates that only one transcript is expressed throughout the soma and the CNS, and that the transcript is highly expressed during early fetal development. We conclude that the delineation of the function of the HOPA gene locus merits further study.

Blotting, Northern↗
PubMedDate not supplied

Synthesis and expression of the gene coding for noxiustoxin, a K+ channel-blocking peptide from the venom of the scorpion Centruroides noxius.

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E, coli vector pCSP 105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

Amino Acid Sequence↗
PubMedDate not supplied

The calcium-binding protein calreticulin is covalently modified in rat liver by a reactive metabolite of the inhalation anesthetic halothane.

A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.1% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.

Amino Acid Sequence↗
PubMedDate not supplied

Serum antibodies from halothane hepatitis patients react with the rat endoplasmic reticulum protein ERp72.

Immunoblotting studies have previously shown that serum antibodies from halothane hepatitis patients react with several liver microsomal proteins that have been modified by the trifluoroacetyl halide metabolite of halothane. In this study, an 80-kDa protein recognized by the patients' antibodies has been purified from rat liver microsomes and characterized. When the purified trifluoroacetylated 80-kDa and native 80-kDa proteins were employed as test antigens in an enzyme-linked immunosorbent assay, serum antibodies from halothane hepatitis patients reacted with both of these proteins to a significantly greater extent than did serum antibodies from control patients. Amino acid sequence analyses of several hydrolytic peptide fragments of the 80-kDa protein showed that the protein was 99% identical to the deduced amino acid sequence of a murine cDNA of the luminal endoplasmic reticulum protein ERp72. These results indicate that trifluoroacetylated ERp72 in the liver of halothane hepatitis patients may induce immune responses against epitopes present on the covalently altered protein and those present on the native protein and may have a role in halothane hepatitis. In addition, immunoblot and immunohistochemical studies revealed that the 80-kDa protein was present in all tissues studied, but was in highest concentration in liver, adipose tissue, ovaries, and testes and was enriched in specific cells of some organs. In the future, these findings should help define the physiological function of ERp72.

Amino Acid Sequence↗
PubMedDate not supplied

[Beta-lactam antibiotic sensitivity in Aeromonas spp. of clinical, animal, and environmental origin].

Susceptibility to beta-lactam antibiotics was investigated in Aeromonas spp. Microorganisms were isolated from both, clinical and water creek samples, as well as from processed raw chicken carcasses. Aeromonas like colonies were identified by means of Aerokey II and API 20 E System (Bio-Merieux). A. hydrophila prevailed both of human origin (44%) and water creek samples (41%), while A. caviae ranked first among raw chicken samples (65%). Dilution testing by Agar Method was performed to determine minimum inhibitory concentration (MIC), following NCCLS standards. All tested microorganisms were susceptible to third generation cephalosporin, cefepime, imipenem, aztreonam, and resistant to ampicillin. Only with cefepime and aztreonam exceptions, strains of human origin showed higher values of MIC90 than environmental ones. These results suggest that antibiotic resistance is mainly due to a steady environmental pressure, on account of the widely used above mentioned compounds.

Aeromonas↗
PubMedDate not supplied

Isozymes of cytochrome P-450 that metabolize naphthalene in liver and lung of untreated mice.

Four isozymes, designated P-450m50a, P-450m50b, P-450m51a, and P-450m51b, have been isolated from liver microsomes of untreated male CD-1 mice. Electrophoretograms revealed that the molecular weights of P-450m51a and P-450m51b were about 51,000, whereas those of P-450m50a and P-450m50b were about 49,700 and 49,500, respectively. The present study is concerned mainly with the elucidation of the characteristics of P-450m50b and P-450m51a, since they appear to be major forms of cytochrome P-450 in mouse liver. End group analyses have revealed that these forms differ not only from P1-450, isolated previously from mouse liver, but also from any other known isozymes of cytochrome P-450. Naphthalene was metabolized by both P-450m50b and P-450m51a, but the turnover number of P-450m50b was 2-4 times greater than that of P-450m51a. Moreover, P-450m50b formed predominantly (1R,2S)-naphthalene 1,2-oxide, as determined by the formation of trans-1(R)-hydroxy-2(R)-glutathionyl-1,2-dihydronaphthalene, whereas P-450m51a formed both (1R,2S)- and (1S,2R)-naphthalene 1,2-oxide in about equal amounts. Although cytochrome P-450c, isolated from rat liver, is also known to convert naphthalene predominantly to (1R,2S)-naphthalene 1,-2-oxide, an antibody prepared against cytochrome P-450c failed to inhibit the formation of the glutathione conjugate when the 1R,2S-oxide was formed by either pure P-450m50b or by microsomes from mouse lung. Thus, P-450m50b probably is not orthologous to cytochrome P-450c.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗