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Biomedical subjects

B M Martin

Publications and source records attributed to B M Martin.

At least 109 records · Page 6Linked to original sources
PubMed1992

Flavobacterium meningosepticum peptide:N-glycosidase: influence of ionic strength on enzymatic activity.

Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium. By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength. We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.

Amidohydrolases↗
PubMed1992

Calretinin distribution in the thalamus of the rat: immunohistochemical and in situ hybridization histochemical analyses.

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei. A subpopulation of calretinin cell bodies was also identified in the reticular nucleus. The mediorostral lateral posterior nucleus, subparafascicular, lateral geniculate and habenular nuclei also contained calretinin messenger RNA probe label. In contrast, no positive cells were found in the anterior, ventral or posterior thalamic nuclei. The distribution of calretinin cells did not correspond directly with that of other histochemical markers. Thus, the in situ hybridization histochemical and immunohistochemical results revealed calretinin as a unique identifying marker for distinct sets of thalamic neurons.

Animals↗
PubMed1992

Type IV collagen synthesis and accumulation in neonatal rat aortic smooth muscle cell cultures.

The production of type IV collagen by cultured neonatal rat aortic smooth muscle cells was monitored over a three-week period to further characterize the extracellular matrix of this unique culture system. Type IV collagen was quantified using a dot immunobinding assay and was found to represent 1% or less of the total collagen produced by these cells in culture. Total collagen represented up to 33% of the total protein. The pattern of type IV collagen production in the media and the cell layer suggests that although these cells synthesize and secrete type IV collagen from the onset of culture, type IV collagen deposition only occurs after the cells have reached confluence. In the presence of ascorbate the amount of type IV collagen peaked in the media in preconfluent cultures. In the absence of ascorbate, little type IV collagen was detected in the media. On the other hand, the presence or absence of ascorbate made little difference in the amount of the total collagen detected in the media, although hydroxylation was affected. Remarkably, in the absence of ascorbate type IV collagen accumulation in the cell layer was similar by the end of the culture period to that in cultures treated with ascorbate. Laminin was not affected by the presence or absence of ascorbate. When these cells were exposed to ascorbate for 24 hours, a peak of soluble elastin was detected in the media. However, soluble elastin was not detected in the media in the absence of ascorbate or in cultures which were maintained in the presence of ascorbate. Modulation of the extracellular matrix with ascorbic acid indicated that type IV collagen deposition did not depend on the presence of ascorbic acid and that there was no discernable interaction between type IV collagen, laminin, and elastin.

Animals↗
PubMed1992

Tyrosine-ester sulfotransferase from rat liver: bacterial expression and identification.

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.

Amino Acid Sequence↗
PubMed1992

IGF-I regulation of elastogenesis: comparison of aortic and lung cells.

Rat neonatal aortic smooth muscle and pulmonary fibroblast cell cultures were exposed to different amounts of insulin-like growth factor-I (IGF-I, 1-100 ng/ml of medium) for 24 h. Aortic smooth muscle cells exhibited an increase in both steady-state levels of tropoelastin mRNA and soluble elastin with increasing amounts of IGF-I, suggesting that the growth factor is acting by increasing transcription or transcript stability. In contrast, pulmonary fibroblast cultures did not exhibit an elastogenic response to IGF-I because neither the steady-state levels of tropoelastin mRNA nor soluble elastin were affected. Transient transfection of the two cell cultures with a chimeric construct containing 500 bp of the elastin gene 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene showed that reporter activity was increased threefold in smooth muscle cells treated with IGF-I, whereas activity remains essentially the same in control and growth factor-treated pulmonary fibroblast cells. Receptor binding analyses revealed that both cell types possess the type I IGF-I receptor. Therefore, the lack of an elastogenic response in the lung cells cannot be attributed to lack of the appropriate receptor. These data, obtained in vitro with cell types that are principal producers of lung and aortic elastin, agree with results obtained in vivo. This agreement suggests that the regulation of elastin gene expression varies among cells derived from different tissues and furthermore provides model systems to investigate differential regulation of the elastin gene.

Animals↗
PubMed1992

Age-related changes in alpha 1- and alpha 2-chain type IV collagen mRNAs in adult mouse glomeruli: competitive PCR.

Studies of age-related changes in glomerular extracellular matrix (ECM) synthesis in normal mice have been hampered by the difficulty of isolating sufficient numbers of intact glomeruli and by the inability to quantify different mRNA species. The purpose of this study was to identify and quantitate the individual mRNAs coding for alpha 1- and alpha 2-chains of type IV collagen in isolated, single glomeruli of normal mice at different ages. These data on normal ECM synthesis were necessary for the understanding of glomerulosclerosis, a condition characterized by excess deposition of collagen. Pools of freshly microdissected adult mouse glomeruli were reverse transcribed in situ, and alpha 1-IV and alpha 2-IV collagen mRNAs were individually amplified by means of specific primers and the polymerase chain reaction (PCR), according to a previously published method. A competitive PCR assay, based on utilization of mutated cDNAs, allowed the reproducible, quantitative, and separate determination of the absolute amounts of both alpha 1-IV and alpha 2-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of alpha 1-IV and alpha 2-IV collagen mRNA were 208 +/- 36.0 x 10(-4) and 161.2 +/- 18.6 x 10(-4) amol/glomerulus in 5-wk-old mice. There were no significant age-related differences at 8, 12, and 24 wk. The mean levels over this period were 60.2 +/- 4.9 x 10(-4) for alpha 1-IV collagen mRNA and 63.9 +/- 5.8 x 10(-4) amol/glomerulus for alpha 2-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endothelial cell growth arrest.

Angiotensin converting enzyme (ACE) is a zinc-containing dipeptidase that converts angiotensin I to angiotensin II, a powerful vasoconstrictor and smooth muscle growth factor. ACE activity has been shown to be dynamically regulated by hormones, ACE inhibitors, and endothelial cell growth state. To study how ACE expression is regulated, we isolated and sequenced the bovine ACE gene using both ACE-specific cDNA and genomic clones. Bovine ACE cDNA encodes a single polypeptide of 1,306 residues with a molecular mass of 150 kd. Bovine ACE is approximately 80% homologous to that of other species. It contains two homologous domains of equal size. Alignment of ACE sequences from bovine, human, mouse, and rabbit reveals that during evolution both domains have been highly conserved. We used the bovine ACE cDNA to study regulation of ACE gene expression during density-dependent growth arrest. As endothelial cells became growth-arrested (6 days after confluence), there was a 12-fold increase in ACE activity and a 90% decrease in DNA synthesis. Immunocytochemically detectable ACE markedly increased in growth-arrested cells. The increase in ACE was due to increased ACE gene expression, as assayed by RNase protection, which showed a 20-fold increase in ACE-specific mRNA. The present study shows that bovine ACE is highly regulated by endothelial cell growth state at the level of protein and mRNA expression. Such dynamic regulation may have important consequences for angiotensin II production during endothelial cell proliferation after arterial injury.

Amino Acid Sequence↗
PubMed1992

Elastin synthesis and accumulation in irradiated smooth muscle cell cultures.

Multilayer cultures of neonatal rat aortic smooth muscle cells, which were actively synthesizing elastin, were exposed to gamma-radiation. Elastin synthesis and accumulation was measured as a function of time after irradiation and compared to control (non-irradiated) cultures. Cells exposed to 50 Gy ceased to divide but continued to synthesize and accumulate elastin. The culture morphology suggested that the irradiated cells accumulated an extensive extracellular matrix between their cell layers. Interestingly, the amount of elastin accumulated in the irradiated cultures was nearly the same as in the controls despite the difference in cell number in the two cultures. Thus, on a per cell basis, the elastin accumulation was greater in the irradiated cultures than in the controls.

Animals↗
PubMed1992

Noxiustoxin and leiurutoxin III, two homologous peptide toxins with binding properties to synaptosomal membrane K+ channels.

Noxiustoxin (NTX), a short-chain peptide toxin from the scorpion C. noxius, binds to a single class of non-interacting binding sites in brain synaptosomal membranes with a KD = 160-300 nM and a Bmax = 2.2 pmols.mg-1 protein. The K+ channel blocking agents tetraethylammonium, 4-aminopyridine and Charybdotoxin partially inhibit the binding of [125I]NTX, while a variety of toxins targeted against Ca2+ and Na+ channels have no effect, suggesting that NTX interacts with a bona fide K+ channel protein. NTX totally displaces the binding of [125I]Leiurutoxin III, a novel peptide from L. quinquestriatus venom with N-terminal amino acid sequence homologous to that of NTX. These results suggest that both toxins bind to a common receptor site and are promising tools to dissect the molecular mechanisms of channel-toxin interaction.

4-Aminopyridine↗
PubMed1991

The effect of beta-aminopropionitrile on elastin gene expression in smooth muscle cell cultures.

When beta-aminopropionitrile (BAPN) is added to neonatal rat aortic smooth muscle cell cultures there is a decrease in insoluble elastin accumulation with a concomitant increase in tropoelastin and tropoelastin fragments in the culture medium. The experiments described here examine the biological significance of this fragmentation. BAPN, as well as purified tropoelastin fragments isolated from spent medium of cells grown in the presence of BAPN, were added to cultures. A decrease in elastin mRNA was observed in cultures grown in the presence of BAPN and also in those cultures to which the purified tropoelastin moieties were added. These studies indicate that the inhibition of lysyl oxidase by BAPN prevents elastin crosslinking which results in an increase in tropoelastin moieties, thus leading to a down regulation of the steady state levels of elastin mRNA.

Aminopropionitrile↗
PubMed1991

A metabolite of halothane covalently binds to an endoplasmic reticulum protein that is highly homologous to phosphatidylinositol-specific phospholipase C-alpha but has no activity.

When the inhalation anesthetic halothane was administered to rats, a 58 kDa protein in the liver became covalently labeled by the trifluoroacetyl chloride metabolite of halothane. The amino acid sequences of the N-terminal and of several internal peptide fragments of the protein were 99% homologous to that of the deduced amino acid sequence of a cDNA reported to correspond to phosphatidylinositol-specific phospholipase C-alpha. The purified trifluoroacetylated 58 kDa protein or native 58 kDa protein, however, did not have phosphatidylinositol-specific phospholipase C activity. We conclude that the reported cDNA of phosphatidylinositol-specific phospholipase C-alpha may encode for a microsomal protein of unknown function.

Amino Acid Sequence↗
PubMed1991

Human placental sialidase complex: characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies.

Sialidase isolated from human placenta is associated with several proteins including acid beta-galactosidase, carboxypeptidase, N-acetyl-alpha-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449-456, 1990) as a sialidase protein: this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (gamma 1).

Amino Acid Sequence↗
PubMed1991

gamma-Aminobutyric acid (GABA) induces a receptor-mediated reduction in GABAA receptor alpha subunit messenger RNAs in embryonic chick neurons in culture.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, is known to interact with a subclass of receptors that activate a ligand-gated chloride ion channel. Exposure of cultured embryonic chick neurons to physiological concentrations of GABA results in a time-dependent down-regulation of these GABAA receptors. To delineate the cellular mechanism(s) responsible for agonist-induced down-regulation of GABAA receptors we quantified the levels of GABAA receptor alpha subunit messenger RNAs, which encode the subunit(s) containing agonist recognition site(s), and observed a marked reduction in alpha subunit mRNAs following exposure of embryonic chick neurons to GABA. Both the down-regulation of GABAA receptors and the reduction in alpha subunit mRNAs induced by GABA were completely antagonized by the specific GABAA receptor antagonist SR-95531. These data demonstrate the presence of an agonist-induced receptor-mediated mechanism for regulating the expression of receptor subunit-encoding mRNAs that may be involved in the development of tolerance to the pharmacological actions of drugs known to act via GABAA receptors.

Animals↗
PubMed1991

Discharge effect on pancreatic exocrine secretion produced by toxins purified from Tityus serrulatus scorpion venom.

Three toxic polypeptides were purified from the venom of the Brazilian scorpion Tityus serrulatus by means of gel filtration in Sephadex G-50 and ion-exchange chromatography in carboxymethylcellulose. The peptides are basic molecules with molecular weights in the range of 7000 for which the amino acid compositions and sequences were determined. The effect of the purified peptides on pancreatic exocrine secretion in the guinea pig was studied. Biochemical measurements show that the cells are stimulated by these peptides to discharge their zymogen granules. Light and electron microscopic images confirm the biochemical measurements. At the light microscope level, acinar cells show dramatically fewer zymogen granules than in control pancreas with the appearance of large vacuoles and some loss of morphological integrity. Electron micrographs display apical regions devoid of zymogen granules and condensing vacuoles whereas acinar lumina contain crystalline secretory material. The secretory effect observed in vitro is comparable to that of carbamylcholine and that of the peptidergic secretagogue cholecystokinin-pancreozymin.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of a human liver carboxylesterase isoenzyme.

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.

Amino Acid Sequence↗
PubMed1991

A controlled precursor add-back model of elastogenesis in smooth muscle cell cultures.

Neonatal rat aortic smooth muscle cell cultures are capable of synthesizing and accumulating relatively large amounts of insoluble elastin in the extracellular matrix. There are two major soluble elastin molecules in these cultures, one of 77 kDa (protropoelastin) and the other of 71 kDa (tropoelastin). We examined the ability of the cell culture system to insolubilize exogenously added soluble elastin precursor moieties into the elastin matrix. To accomplish this, cultures were allowed to develop an enriched elastic fiber matrix for approximately two weeks in first passage. This accumulated matrix then served as the "substrate" for the exogenously added precursor elastin molecules. Culture-derived radioactive soluble elastin was added to the "substrate" cultures and the presence of radioactivity in the insoluble elastin as well as in the lysine-derived crosslinks unique to elastin (desmosines) was measured. When purified [3H]-valine radiolabeled protropoelastin was used, more than 15% of the radioactivity added was detected in the alkali-resistant insoluble elastin within 24 hours. After an initial 4-hour incubation of the cells with [3H]-lysine-labelled soluble elastin, most of the radioactivity in the insoluble elastin was associated with the lysine and only a negligible amount was detected in the desmosines. However, during a 16-day chase period, the ratio of radioactive desmosines to lysine increased dramatically, suggesting that not only insolubilization, but crosslinking occurs as well. The add-back system described herein should provide a means to probe the molecular properties of protropoelastin and increase our understanding of the mechanisms of elastic fiber formation.

Animals↗
PubMed1991

RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney.

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.

Animals↗