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Biomedical subjects

M L Kahn

Publications and source records attributed to M L Kahn.

At least 19 recordsLinked to original sources
PubMed1999

Glycoprotein V-deficient platelets have undiminished thrombin responsiveness and Do not exhibit a Bernard-Soulier phenotype.

Adhesion of platelets to extracellular matrix via von Willebrand factor (vWF) and activation of platelets by thrombin are critical steps in hemostasis. Glycoprotein (GP) V is a component of the GPIb-V-IX complex, the platelet receptor for vWF. GPV is also cleaved by thrombin. Deficiency of GPIb or GPIX results in Bernard-Soulier syndrome (BSS), a bleeding disorder in which platelets are giant and have multiple functional defects. Whether GPV-deficiency might also cause BSS is unknown as are the roles of GPV in platelet-vWF interaction and thrombin signaling. We report that GPV-deficient mice developed normally, had no evidence of spontaneous bleeding, and had tail bleeding times that were not prolonged compared with wild-type mice. GPV-deficient platelets were normal in size and structure as assessed by flow cytometry and electron microscopy. GPV-deficient and wild-type platelets were indistinguishable in botrocetin-mediated platelet agglutination and in their ability to adhere to mouse vWF A1 domain. Platelet aggregation and ATP secretion in response to low and high concentrations of thrombin were not decreased in GPV-deficient platelets compared with wild-type. Our results show that (1) GPV is not necessary for GPIb expression and function in platelets and that GPV deficiency is not likely to be a cause of human BSS and (2) GPV is not necessary for robust thrombin signaling. Whether redundancy accounts for the lack of phenotype of GPV-deficiency or whether GPV serves subtle or as yet unprobed functions in platelets or other cells remains to be determined.

Animals↗
PubMed1999

Distribution of two isoforms of NADP-dependent isocitrate dehydrogenase in soybean (Glycine max).

Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized. The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other. Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively. Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated. Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein. IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues. Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean.

Antibody Specificity↗
PubMed1999

Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides.

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0. 2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.

Chromatography, High Pressure Liquid↗
PubMed1999

Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin.

Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.

Adenosine Triphosphate↗
PubMed1998

Gene and locus structure and chromosomal localization of the protease-activated receptor gene family.

Protease-activate receptors (PARs) mediate activation of platelets and other cells by thrombin and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis, and other normal and pathological processes. We report here the structure of the mouse and human PAR3 genes as well as the organization of a PAR gene cluster encompassing the genes encoding PARs 1, 2, and 3. We also report the structure of the mouse and human PAR4 genes, which map to distinct chromosomal locations and encode a new thrombin receptor. PARs 1-4 are all encoded by genes with the same two exon structure. In each case, exon 1 encodes a signal peptide, and exon 2 encodes the mature receptor protein. These are separated by an intron of variable size. The genes encoding PARs 1-3 all map to chromosome 13D2 in mouse and chromosome 5q13 in human. In mouse, all three genes are located within 80 kilobases of each other. The PAR1 gene is located centrally and is flanked upstream by the PAR3 gene and downstream by the PAR2 gene in both species. The proximity of the PAR1 and PAR3 genes suggests the possibility that these genes might share regulatory elements. A comparison of the structures of the PAR amino acid sequences, gene structures, locus organization, and chromosomal locations suggests a working model for PAR gene evolution.

Animals↗
PubMed1998

Generation of up-regulated allosteric variants of potato ADP-glucose pyrophosphorylase by reversion genetics.

Mutagenesis of the large subunit (LS) of the potato ADP-glucose pyrophosphorylase generated an enzyme, P52L, that was insensitive to 3-phosphoglycerate (3-PGA). To identify additional residues involved in 3-PGA interaction, we subjected P52L LS DNA to a second round of mutagenesis and identified second-site revertants by their ability to restore glycogen accumulation as assessed by iodine (I2) staining. Enzymes from class I revertants with normal I2-staining had an 11- to 49-fold greater affinity for the activator 3-PGA compared with the P52L mutant and a decreased sensitivity to the inhibitor orthophosphate. Sequence analysis of these class I revertants identified a P66L mutation in R4, an E38K mutation in R20, and a G101N mutation in R10 and R32. These mutations appear to restore 3-PGA binding by counteracting the effect of the P52L mutation because introducing E38K or G101N into the wild-type LS led to enzyme variants with higher affinity for the activator 3-PGA and increased resistance to the inhibitor orthophosphate. The generation of these revertant enzymes provides additional structure-function information on the allosteric regulation of higher plant ADP-glucose pyrophosphorylases and validates a strategy for developing novel variants of the enzyme that may be useful in manipulating starch biosynthesis in higher plants.

Allosteric Site↗
PubMed1998

A dual thrombin receptor system for platelet activation.

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.

Amino Acid Sequence↗
PubMed1997

Protease-activated receptor 3 is a second thrombin receptor in humans.

Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled receptor. Knockout of the gene encoding this receptor provided definitive evidence for a second thrombin receptor in mouse platelets and for tissue-specific roles for different thrombin receptors. We now report the cloning and characterization of a new human thrombin receptor, designated protease-activated receptor 3 (PAR3). PAR3 can mediate thrombin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to thrombin.

Amino Acid Sequence↗
PubMed1997

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.

Amino Acid Sequence↗
PubMed1996

Role of the thrombin receptor in development and evidence for a second receptor.

Thrombin, a coagulation protease generated at sites of vascular injury, activates platelets, endothelial cells, leukocytes and mesenchymal cells. A G-protein-coupled receptor that is proteolytically activated by thrombin is a target for drug development aimed at blocking thrombosis, inflammation and proliferation. Here we show that although disruption of the thrombin receptor (tr) gene in mice causes about half of the tr-/- embryos to die at embryonic day 9-10, half survive to become grossly normal adult mice with no bleeding diathesis. Strikingly, tr-/- platelets respond strongly to thrombin, whereas tr-/- fibroblasts lose their ability to respond to thrombin. We conclude that the thrombin receptor plays an unexpected role in embryonic development, suggesting a possible new function for the 'coagulation' proteases themselves. Moreover, a second platelet thrombin receptor exists, and different thrombin receptors have tissue-specific roles. This may allow development of therapeutics that will selectively block thrombin's different cellular actions.

Adenosine Triphosphate↗
PubMed1996

Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase.

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.

Amino Acid Sequence↗
PubMed1996

Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.

Allosteric Regulation↗
PubMed1996

Experience with the P.A.S.-PORT venous access device in patients with gynecologic malignancies.

Experience with the P.A.S.-PORT, a peripherally implanted central venous access device, is evaluated in a retrospective review of 154 patients from July 1991 to June 1994. Blood could not be aspirated from six patients. Complications included temporary minor thrombophlebitis in seven patients (4.5%), symptomatic axillary or subclavian vein thrombosis in five patients (3.2%), clotted port in two patients (1.2%), port pocket cellulitis in two patients (1.2%), and fungal sepsis in two patients (1.2%). In six patients (3.8%) the P.A.S.-PORT had to be removed because of complications. The P.A.S.-PORT facilitated delivery of chemotherapy, parenteral nutrition, blood products, antibiotics, hydration, and blood sampling. It was demonstrated that the P.A.S.-PORT may be inserted and used with a low incidence of complications in gynecologic cancer patients.

Anti-Bacterial Agents↗
PubMed1995

ADP-ribosylation of Rhizobium meliloti glutamine synthetase III in vivo.

The control of glutamine synthetase (GS), the first enzyme in the main pathway used by Rhizobium meliloti to assimilate ammonia, is central to cellular nitrogen metabolism. R. meliloti is unusual in having three distinct types of GS, including a unique GS, GSIII, that differs considerably from both GSI, which resembles other bacterial GS proteins and GSII, which resembles the GS found in eukaryotes. We show here that GSIII can be post-translationally modified in vivo by ADP-ribosylation at an arginine residue. 32PO4 attached to GSIII during bacterial growth as part of the modifying group could be removed by treatment with snake venom phosphodiesterase or by turkey erythrocyte ADP-ribosylarginine hydrolase. Treatment of modified GSIII with hydroxylamine at neutral pH releases a chromophore that has the retention time of ADP-ribose when analyzed by reversed-phase high performance liquid chromatography. ADP-ribosylation inhibits GSIII activity.

Adenosine Diphosphate Ribose↗
PubMed1994

Effects of organic acids and low pH on Rhizobium meliloti 104A14.

In the symbiotic relationship between Rhizobium meliloti and alfalfa (Medicago sativa), the bacteria are enclosed within the plant cell by a membrane that may function like a plant vacuolar membrane and maintain a pH between 5.5 and 6.0. Free-living Rhizobium meliloti 104A14 is sensitive to pH in this range and its sensitivity was influenced by the presence of acetate and other monocarboxylic acids. R. meliloti can grow at pH 6.0 in 3 mM succinate but does not grow at pH 6.2 if 10 mM acetate is added. The combination of low pH and acetate is bacteriostatic. Measurement of internal pH (pHi) using 14C-labelled benzoate as a permeant acid showed that growth inhibition occurs when pHi falls below 7.15.

Acetates↗
PubMed1993

Isolation and characterization of a novel glutamine synthetase from Rhizobium meliloti.

Two glutamine synthetases, GSI and GSII, are found in most rhizobia. However, WSU650, a Rhizobium meliloti glnA glnII mutant that lacks both enzymes, can grow without a glutamine supplement in minimal medium that contains both ammonium and glutamate. The bacteria contained a third glutamine synthetase, GSIII, which has been purified and partially characterized. GSIII had considerable glutamine synthetase activity when assayed using a semibiosynthetic (glutamate- and hydroxylamine-dependent) assay, but had no detectable transferase (glutamine- and hydroxyl-amine-dependent) activity. GSIII was inhibited by ADP and pyrophosphate but not by various nitrogen-containing metabolites that inhibit other GS enzymes. Activity was also inhibited by methionine sulfoximine, a transition state analog, but the concentration needed to inhibit GSIII was 50 to 100 times higher than that needed to inhibit GSI or GSII. GSIII had a Km for glutamate of 13.3 mM, for ammonium of 33 mM, and for hydroxylamine of 5.3 mM with a pH optimum of 6.8 and a temperature optimum of 50 degrees C. The purified protein had related subunits of 46.5 and 49 kDa and a native molecular mass of 355 kDa, indicating the native enzyme was an octamer. Polyclonal antibodies specific for GSIII reacted with a protein of similar molecular weight in Escherichia coli strains that carry R. meliloti glnT on a plasmid. GSIII activity was detected in some of these strains that contained glnT. Extracts of root nodules formed by WSU650 also react with the antibodies.

Aspartate-Ammonia Ligase↗
PubMed1993

Isolation and characterization of a cDNA encoding NADP(+)-specific isocitrate dehydrogenase from soybean (Glycine max).

A cDNA that encodes an NADP-specific isocitrate dehydrogenase (IDH) was cloned from a soybean nodule cDNA library by complementation of an Escherichia coli mutant that lacked IDH. DNA sequence analysis showed that the 1583 bp soybean cDNA could encode a protein that shares 63.9% amino acid sequence identity with the Saccharomyces cerevisiae NADP-IDH and long sequences of identity to an IDH from pig. Southern blot analysis suggests that this gene corresponds to a gene family made up of no more than two loci. The IDH cDNA hybridized to a 1.7 kb soybean mRNA and the relative amount of this transcript in soybean leaves, nodules and roots was 1:3.4:7.7. In alfalfa, a 1.7 kb mRNA was also found but the ratios for the corresponding tissues were 1:7.4:7.7. IDH activity was detected in the complemented E. coli strain and the electrophoretic mobility of this activity in nondenaturing polyacrylamide gels was identical to that of an IDH in extracts from soybean cotyledons or nodule cytosol. NADP-IDH specific activity in the E. coli host strain varied with growth phase; the highest rates (ca. 180 nmol/min per mg protein) were observed in late-stationary-phase cells. The enzyme had a broad pH optimum of 8.0 to 9.5 and had an absolute metal cofactor requirement, preferring Mn2+ below pH 8.0 and Mg2+ above pH 8.0. The Km for isocitrate and NADP was 21 microM and 11 microM respectively with Mn2+ as cofactor and 13 microM and 12 microM with Mg2+ as cofactor.

Amino Acid Sequence↗