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B M Martin

Publications and source records attributed to B M Martin.

At least 127 records · Page 7Linked to original sources
PubMed1991

Prolonged ethanol inhalation decreases gamma-aminobutyric acidA receptor alpha subunit mRNAs in the rat cerebral cortex.

Ethanol administration to rats by ethanol vapor inhalation (14 days) results in a 40-50% reduction in the level of gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit mRNAs [4.4 and 4.8 kilobases (kb)] in the cerebral cortex. The level of alpha 2 subunit mRNA (8.0 kb) was also reduced by 29%, whereas there was no effect of prolonged ethanol exposure on the level of alpha 3 subunit mRNA (3.1 kb). Ethanol exposure did not alter the steady state levels of cerebral cortical glutamic acid decarboxylase or beta-actin mRNAs. Moreover, no alterations in the levels of total RNA, poly(A)+ RNA, or rRNA were observed, suggesting that the ethanol-induced reductions in GABAA receptor alpha 1 and alpha 2 subunit mRNAs were not the result of a generalized effect of ethanol administration on transcription or mRNA turnover. These ethanol-induced reductions in GABAA receptor alpha subunit mRNAs may underlie alterations in GABAA receptor function or number observed following prolonged ethanol exposure in rats.

Administration, Inhalation↗
PubMed1991

Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain.

A fragment of the cDNA encoding the third intracellular loop of the rat m1 muscarinic receptor was cloned, and the DNA was expressed in Escherichia coli as a fusion protein. The fusion protein was purified and utilized as an antigen to raise a polyclonal antiserum in rabbits. Chinese hamster ovary cells stably transfected with the cDNA encoding each of the five known subtypes of muscarinic receptor were used as tissue sources to test the antiserum. The antiserum was found to quantitatively immunoprecipitate m1 muscarinic receptors, while not precipitating m2, m3, m4, or m5 receptors. This selective antiserum was utilized to quantify the density of m1 muscarinic receptors in seven selected areas of the rat brain. Thus, cortex was found to contain approximately 0.8 pmol/mg of membrane protein, which represents 34% of the total density of muscarinic receptors. Similarly, hippocampus (1 pmol/mg; 47%), striatum (0.8 pmol/mg; 29%), and olfactory tubercule (0.9 pmol/mg; 35%) are rich in m1 receptors. In contrast, thalamus/hypothalamus contained only 0.15 pmol/mg, representing approximately 16% of the total density of muscarinic receptors, whereas pons/medulla (0.03 pmol/mg; 5%) and cerebellum (less than 0.01 pmol/mg; 2%) had very low levels of expression of m1 receptors. The development of a selective antiserum has provided a means for the quantification of a specific subtype of muscarinic receptor in tissues, such as the brain, that express multiple subtypes. This methodology will be applicable not only to the other subtypes of muscarinic receptor but also to the subtypes of several other neurotransmitter receptors that lack selective drugs with which to study them.

Animals↗
PubMed1990

Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs.

In this paper we report the amino acid sequence of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus as determined from the nucleotide sequence of the PPDK gene. Comparison of the B. symbiosus PPDK amino acid sequence with that of the maize PPDK [Matsuoka, M., Ozeki, Y., Yamamoto, N., Hirano, H., Kamo-Murakami, Y., & Tanaka, Y. (1988) J. Biol. Chem. 263, 11080] revealed long stretches of homologous sequence (greater than 70% identity), which contributed to an overall sequence identity of 53%. The circular dichrosim spectra, hydropathy profiles, and calculated secondary structural elements of the two dikinases suggest that they may have very similar tertiary structures as well. A comparison made between the amino acid sequence of the maize and B. symbiosus dikinase with other known protein sequences revealed homology, concentrated in three stretches of sequences, to a mechanistically related enzyme, enzyme I of the Escherichia coli PEP: sugar phosphotransferase system [Saffen, D. W., Presper, K. A., Doering, T. L., Roseman, S. (1987) J. Biol. Chem. 262, 16241]. It is proposed that (i) these three stretches of sequence constitute the site for PEP binding and catalysis and a possible site for the regulation of enzymatic activity and (ii) the conserved sequences exist in a third mechanistically related enzyme, PEP synthase.

Amino Acid Sequence↗
PubMed1990

Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods.

The cloning and characterization of cDNAs for the catalytic subunit of calcineurin (CN) from murine and human brain libraries were carried out using nonisotopic methods. A murine cDNA clone encoding a protein of 521 amino acids (Mr approximately 58,650) was isolated; overlapping clones established a 3'-untranslated region of 554 base pairs preceding the poly(A) tail. Homologous cDNAs from human brain showed greater than 92% nucleotide sequence identity in both coding and non-coding regions with greater than 99% conservation of amino acid sequence. A second class of cDNAs lacking a specific 30-base pair region following the calmodulin-binding domain was found in four murine and human libraries. Oligonucleotide probes for both cDNA isoforms hybridized to mRNA from several brain regions indicating the existence of transcripts in vivo. The nucleotide sequences of the two forms were identical except for the inserted sequence, and Southern blot analysis of mouse and rat DNA was consistent with their having originated from the same gene; these data suggest that alternative splicing may give rise to molecular isoforms of the catalytic subunit in brain. Northern blots showed a predominant mRNA for CN in most tissues of approximately 4.0 kilobases (kb) with lower amounts of a 3.6-kb species. Brain showed 10 times more of these mRNAs than skeletal muscle while other tissues had less than or equal to 5% that in brain. In testis, multiple mRNAs were observed, with the major forms being approximately 2.8 and 1.6 kb; the total amount of CN message was about 15% that in brain. The presence of mRNA isoforms of the catalytic subunit may provide for isoenzymes of this phosphatase having distinct phosphoprotein substrate specificities or regulatory properties. The structural relatedness of CN to other mammalian serine/threonine protein phosphatases was highest over a region of approximately 240 amino acids near the amino terminus of this subunit, with greater similarity to protein phosphatase 2A than protein phosphatase 1. The conservation of many regions found in lambda phage phosphatase (Cohen, P.T.W., and Cohen, P. (1989) Biochem. J. 260, 931-934) indicates a common origin for the catalytic domain of this enzyme.

Amino Acid Sequence↗
PubMed1990

Metabolism-based covalent bonding of the heme prosthetic group to its apoprotein during the reductive debromination of BrCCl3 by myoglobin.

The reductive metabolism of BrCCl3 by ferrous myoglobin leads to the alteration of the prosthetic heme to form products that can be dissociated from the protein and to those that are irreversibly bound to the protein. The major dissociable or soluble heme metabolites have recently been characterized. In this study, the irreversibly bound heme product was characterized by Edman degradation, amino acid analysis, and electronic absorption and mass spectrometry of peptides derived from the altered protein. It was found that the prosthetic heme was modified by a CCl2 moiety derived from BrCCl3 and was covalently bound to histidine residue 93, the normal proximal ligand to the heme-iron. The data are consistent with a mechanism by which the trichloromethyl radical reacts with the heme to form an intermediate that either can alkylate the proximal histidine residue or form soluble metabolites. The covalent bonding of the heme prosthetic moiety to the apoprotein likely leads to a change in the tertiary structure of the protein that may be responsible for its altered catalytic activity as well as its enhanced susceptibility to proteolysis. Similar processes may account, at least in part, for the covalent alteration of the heme prosthetic group of other hemoproteins caused by xenobiotics and endogenous substrates.

Amino Acid Sequence↗
PubMed1990

Genomic DNA 5' to the mouse and human angiotensin-converting enzyme genes contains two distinct regions of conserved sequence.

Genomic DNA 5' to the mouse and human genes encoding angiotensin-converting enzyme has been isolated and analyzed. A sequence comparison identifies two discrete regions of genomic DNA that are highly conserved. One region, found immediately 5' to the mouse and human ACE genes, is a GC rich segment that contains a "TATA box" and several potential Spl binding sites. The second conserved region is found further 5' and contains several potential regulatory cis elements including a possible glucocorticoid responsive element. These two regions of genomic DNA may influence the rate of mRNA transcription from the angiotensin-converting enzyme gene.

Animals↗
PubMed1990

A latent Mr 94,000 gelatin-degrading metalloprotease induced during differentiation of HL-60 promyelocytic leukemia cells: a member of the collagenase family of enzymes.

The treatment of HL-60 promyelocytic leukemia cells with phorbol esters (12-O-tetradecanoylphorbol-13-acetate) results in the appearance of cell substrate adhesion and the release of a Mr 94,000 gelatin-degrading metalloprotease. The appearance of the metalloprotease in the culture medium directly correlates with the timing and extent of cell substrate adhesion over a 24-h period. Anti-Mr 94,000 metalloprotease blocking antibodies were unable to interfere with the HL-60 cell substrate adhesion induced by 12-O-tetradecanoylphorbol-13-acetate, although they were able to specifically remove the Mr 94,000 gelatin-degrading activity from either HL-60 or U-937 cell-conditioned medium. A purified metalloprotease preparation was found to be predominantly latent and activated by organomercurials, acid treatment (pH 2 to 3.6), or 8 M urea. The activating effect of the latter two denaturing treatments suggests that conformational changes may be the common activating mechanism. The different treatments also caused the appearance of lower molecular weight gelatin-degrading bands (in gelatin zymogram gels) in a manner consistent with the autocatalytic cleavage that occurs with other collagnase proenzymes during activation. Edman degradation of a cyanogen bromide fragment from the Mr 94,000 metalloprotease provided the amino acid sequence [PR(C)GVPD] which is present in type I collagenase, stromelysin, and transin proenzyme sequences and partially conserved (V----N substitution) in the type IV collagenase proenzyme. This sequence has been reported to be important in the maintenance of the latent state of the transin proenzyme (R. Sanchez-Lopez et al., J. Biol. Chem., 263: 1892-11899, 1988) and is a sequence unique to collagenase proenzymes. The N-terminal sequence of the Mr 94,000 metalloprotease (AP-QDQST) is unique and distinct from other collagenases. Thus, the Mr 94,000 metalloprotease from HL-60 cells appears to be a distinct and new member of the collagenase family of proteases.

Base Sequence↗
PubMed1990

A novel high-density lipoprotein particle and associated protein in rat plasma.

This report describes the characterization of a novel rat apolipoprotein, which, as partial sequencing suggests, does not correspond to any described protein. The protein (termed PX) has an estimated molecular mass of 19.5 kDa and pI in the range 5.5-5.8. Monoclonal antibodies were obtained against protein PX and results on distribution among rat lipoproteins show it to be associated mainly with high-density lipoproteins (HDL), but also with VLDL. Immunoaffinity chromatography of total HDL shows protein PX to be included in a distinct lipoprotein particle, particularly enriched in free cholesterol, with which only traces of other apolipoproteins are associated. Immunologically crossreacting entities are found in the plasma of several species, including man. Retention of the epitope carried by the protein PX would suggest that it is of particular structural or functional importance. It remains to be established whether its function is associated with lipid metabolism.

Animals↗
PubMed1990

Monocyte activation by smooth muscle cell-derived matrices.

Mononuclear phagocytes adhere to and penetrate the vessel wall endothelium and contact the subendothelial space prior to the development of the atherosclerotic plaque. In an attempt to model the early events of plaque development we used an elastin-rich, multicomponent, cell-derived matrix from neonatal rat aortic smooth muscle cells as a substratum for monocytes. Using this model, we show that human monocyte morphology and metabolism are markedly altered by the matrix substratum. When a mixed mononuclear cell population is seeded on matrix or plastic, only monocytes adhere to the matrix surface. In contrast, lymphocytes as well as monocytes adhere to the plastic surface. The matrix-adherent monocytes develop large intracellular granules and form extensive clusters of individual cells. Metabolically, these cells develop sodium fluoride resistant non-specific esterase activity and their media contain more growth factor activity and PGE2. Although total protein synthesis is equivalent in both cultures, the matrix contact induces an increase in specific proteins in the media. We also show that a purified alpha-elastin substratum induces some, but not all, of the monocyte changes seen when using the matrix substratum. Using the alpha-elastin substratum, there is selective adhesion of monocytes and increased growth factor activity, however, the cells are morphologically different from the matrix-adherent cells. Thus, the use of the smooth muscle cell-derived matrix, in conjunction with purified matrix components, serves as a model that can provide insight into the mechanisms of monocyte adhesion and stimulation by the matrix environment that exists in vivo. Such mechanisms may be particularly important in atherogenesis.

Animals↗
PubMed1990

Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum (Mexican beaded lizard) venom.

A protein toxic to mice was isolated from the venom of the Mexican beaded lizard Heloderma horridum horridum by a combination of gel filtration (Sephadex G-75) and ion exchange chromatography (both diethylaminoethyl-cellulose [DE-cellulose] and carboxymethyl-cellulose [CM-cellulose]). The purified polypeptide component has an apparent mol. wt of 25,500 and is composed of approximately 220 amino acid residues. It has an isoelectric point (pI) of 6.8 and its N-terminal amino acid sequence was shown to be: Glu-Ala-Ser-Pro-Lys-Leu-Pro-Gly-Leu-Met-Thr-Ser-Asn-Pro-Asp-Gln-Gln-Thr- Glu-Ile. The sequence has no significant similarity with any other protein previously reported in the literature. Enzymatic activities such as phospholipase, hyaluronidase and proteinase, commonly present in venoms, could not be demonstrated in this protein. Patch-clamp experiments conducted with excitable membranes show no effects on Na+, K+ or Ca2+ ion channels. Among the constant physiological effects observed in mice injected with this toxin are lethargy, partial paralysis of rear limbs and lowering of body temperature, suggesting that it might be a hypothermic toxin. We propose calling this toxin Helothermine.

Amino Acid Sequence↗
PubMed1990

Isolation, characterization, and mapping of a human acid beta-galactosidase cDNA.

A lambda gt11 human testicular cDNA library was screened with degenerate oligonucleotide probe mixtures based on amino acid sequence data generated from cyanogen bromide fragments and tryptic fragments of purified human beta-galactosidase. Six positive clones were identified after screening 2 x 10(6) plaques. The sequences of these six clones were determined and found to be derived from two different cDNAs. The sequence of the longest of these cDNAs is nearly identical to that recently determined by Oshima et al. (1988). It codes for a 76-kD protein and all 11 peptides that were generated from the purified enzyme. The second clone is shorter by 393 bp in the central portion of the coding region. Analysis by Northern blotting revealed the presence of a single mRNA species of 2.45 kb in lymphoblasts and testicular tissue. It is deduced from the amino acid sequence data that proteolytic processing of the precursor form of beta-galactosidase must occur by cleavage in the carboxy-terminal portion of the polypeptide perhaps around amino acid 530 at a uniquely hydrophilic sequence. Using a probe generated from the 3' region of the cDNA, we have mapped the locus coding for human beta-galactosidase to chromosome 3p21-3pter.

Amino Acid Sequence↗
PubMed1990

Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene.

Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptidyl carboxypeptidase that catalyzes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II. By analyzing cDNA and genomic DNA, we have constructed a consensus sequence encoding the testis isozyme of mouse ACE. Testis ACE cDNA contains 2,435 base pairs and encodes a protein of 732 amino acids. The N-terminal 66 amino acids are unique to the testis isozyme, while the remaining 666 are identical to the carboxyl half of mouse somatic ACE. The overall conservation of amino acid sequence between the testis isozymes of the mouse, rabbit, and human is 78 to 84%. The conservation of amino acids for the N-terminal domain uniquely expressed within the testis is 63 to 67% between these species. Primer extension and RNase protection experiments show that RNA transcription of the testis ACE isozyme begins 16 or 17 bases upstream from the translation start site. A sequence element resembling a TATA box is found 25 bases 5' of the transcription start site. To create its unique isozyme of ACE, the testis begins mRNA transcription in the middle of the exonic-intronic structure of somatic ACE, within a sequence treated as an intron by somatic tissues. Testis ACE is not the result of alternative RNA splicing but seems due to the start of transcription at a unique site within the ACE gene.

Amino Acid Sequence↗
PubMed1990

Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction.

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

Aldehyde Reductase↗
PubMed1989

Identification of the site on calcineurin phosphorylated by Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain.

The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) incorporating approximately 1 mol of phosphoryl group/mol of catalytic subunit, in agreement with a value previously reported (Hashimoto et al., 1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide fractionation using reverse-phase high-performance liquid chromatography yielded a single labeled peptide that contained a phosphoserine residue. Microsequencing of the peptide allowed both the determination of the cleavage cycle that released [32P]phosphoserine and the identity of amino acids adjacent to it. Comparison of this sequence with the sequences of methionyl peptides deduced from the cDNA structure of CN (Kincaid et al., 1988) allowed the phosphorylated serine to be uniquely identified. Interestingly, the phosphoserine exists in the sequence Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the putative CaM-binding site. The phosphorylated serine residue was resistant to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could be powerfully stimulated by effectors of CN phosphatase activity. The mechanism of dephosphorylation may be intramolecular since the initial rate was the same at phosphoCN concentrations of 2.5-250 nM.

Amino Acid Sequence↗
PubMed1989

Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain.

GABAA receptor alpha subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32P-labelled antisense RNA probes derived from human alpha 1 subunit cDNAs. These alpha subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor alpha 1 subunit probes encoding the putative extracellular domain detect at least five alpha subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor alpha subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These alpha subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Animals↗
PubMed1989

Mouse angiotensin-converting enzyme is a protein composed of two homologous domains.

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts angiotensin I into the potent vasoconstrictor angiotensin II. We have used cDNA and genomic sequences to assemble a composite cDNA, ACE.315, encoding the entire amino acid sequence of mouse converting enzyme. ACE.315 contains 4838 base pairs and encodes a protein of 1278 amino acids (147.4 kDa) after removal of a 34-amino acid signal peptide. Within the protein, there are two large areas of homologous sequence, each containing a potential Zn-binding region and catalytic site. These homologous regions are approximately half the size of the whole ACE protein and suggest that the modern ACE gene is the duplicated product of a precursor gene. Mouse ACE is 83% homologous to human ACE in both nucleic acid and amino acid sequence, and like human ACE, contains a hydrophobic region in the carboxyl terminus that probably anchors the enzyme to the cell membrane (Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G., and Corvol, P. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9386-9390). Northern analysis of mouse kidney, lung, and testis RNA demonstrates that the testicular isozyme of ACE is encoded by a single, smaller RNA (2500 bases) than the two message sizes found in kidney or lung (4900 and 4150 bases), and that this testicular RNA hybridizes to the 3' portion of ACE.315.

Amino Acid Sequence↗
PubMed1989

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin.

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.

Amino Acids↗