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M K Jain

Publications and source records attributed to M K Jain.

At least 55 records · Page 3Linked to original sources
PubMed1999

Group I secreted PLA2 and arachidonic acid metabolites in the maintenance of cat LES tone.

Spontaneous tone of in vitro lower esophageal sphincter (LES) circular muscle is associated with elevated levels of arachidonic acid (AA), PGF(2alpha), and increased [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Gq-, Gi3-, and G(i1/i2)-like G proteins. Tone and AA levels were reduced by inhibitors of a pancreatic-like (group I) secreted phospholipase A2 (sPLA2), by the cyclooxygenase inhibitor indomethacin, and by the thromboxane A2 antagonist SQ-29548. In addition, pertussis toxin (PTX) reduced LES tone, confirming a role of PTX-sensitive G proteins in maintenance of LES tone. PGF(2alpha) contracted LES smooth muscle (strips and cells) and increased [35S]GTPgammaS binding to Gq and Gi3 in solubilized LES circular muscle membranes. PGF(2alpha)-induced contraction of LES permeable muscle cells was inhibited by Gq and Gi3 but not by G(i1/i2) and Go antibodies. The thromboxane A2 analog U-46619 contracted LES smooth muscle and increased Gq binding. U-46619-induced contraction was inhibited by Gq but not by Gi3, G(i1/i2), and Go antibodies. LES tone and [(35)S]GTPgammaS binding were significantly reduced by indomethacin. We conclude that group I sPLA2 may mediate "spontaneous" LES tone by producing AA, which is metabolized to PGF(2alpha) and thromboxane A2. These AA metabolites activate receptors linked to Gi3 and Gq to maintain LES contraction.

Animals↗
PubMed1998

Cecropins induce the hyperosmotic stress response in Escherichia coli.

Cecropin A and B, below or near their minimum inhibitory concentrations in viable Escherichia coli, interfered with the rapid NaCl-induced hyperosmotic shrinkage of the cytoplasmic volume (plasmolysis), and also activated the promoter of the hyperosmotic stress gene osmY. The same promoter was also expressed by hyperosmolar NaCl or sucrose, two of the most commonly used antimicrobial food preservatives. Stress responses were monitored during the logarithmic growth phase of E. coli strains that contain specific promoters fused to a luxCDABE operon on a plasmid. The luminescence assay, developed to monitor the transcriptional response to stresses, is based on the premise that organisms often respond and adapt to sublethal environmental adversities by increased expression of stress proteins to restore homeostasis. The luminescence response from these fusion strains to a specific stress occurs as the transcription at the promoter site is activated. Cecropins induced luminescence response only from the osmY-luxCDABE fusion, but not the corresponding stress promoter activation associated with macromolecular or oxidative damage, or leakage of the cytoplasmic content including the proton gradient. The inhibitory effect of cecropins on plasmolysis is interpreted to suggest that the primary locus of action of these antimicrobial peptides in the periplasmic space is on the coupling between the inner and outer membrane.

Amino Acid Sequence↗
PubMed1998

Embryonic expression suggests an important role for CRP2/SmLIM in the developing cardiovascular system.

Proteins of the LIM family are critical regulators of development and differentiation in various cell types. We have described the cloning of cysteine-rich protein 2/smooth muscle LIM protein (CRP2/SmLIM), a LIM-only protein expressed in differentiated vascular smooth muscle cells. As a first step toward understanding the potential functions of CRP2/SmLIM, we analyzed its expression after gastrulation in developing mice and compared the expression of CRP2/SmLIM with that of the other 2 members of the CRP subclass, CRP1 and CRP3/MLP. In situ hybridization in whole-mount and sectioned embryos showed that CRP2/SmLIM was expressed in the sinus venosus and the 2 cardiac chambers at embryonic day 9. Vascular expression of CRP2/SmLIM was first seen at embryonic day 10. At subsequent time points, CRP2/SmLIM expression decreased in the heart but remained high in the vasculature. CRP1 was expressed both in vascular and nonvascular tissues containing smooth muscle cells, whereas CRP3/MLP was expressed only in tissues containing striated muscle. These patterns of expression were maintained in the adult animal and suggest an important role for this gene family in the development of smooth and striated muscle.

Age Factors↗
PubMed1998

Catalytic significance of the specificity of divalent cations as KS* and kcat* cofactors for secreted phospholipase A2.

Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant for the interfacial binary (E*C) and ternary (E*CL) complexes of PLA2 and substrate mimics (L). Of the cations(II) that promote the binding of a mimic to the enzyme at the interface (E*), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary E*CL complexes with kcat* of <1 s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second 18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of E*C and E*CL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn. These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that the higher ground state stability of the E*CS complex with the inhibitory cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen facilitates the near-attack geometry in the E*CaS, and the His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.

Animals↗
PubMed1998

Calcium-triggered selective intermembrane exchange of phospholipids by the lung surfactant protein SP-A.

It is shown that human lung surfactant protein (SP-A) mediates selective exchange of phospholipid probes with unlabeled phospholipid in excess vesicles in the presence of calcium and NaCl. The exchange occurs without leakage of vesicle contents, or transbilayer movement (flip-flop) of the phospholipid probes, or fusion of vesicles. Individual steps preceding the exchange are dissected by a combination of protocols, and the results are operationally interpreted in terms of a model where a calcium-dependent change in SP-A triggers aggregation of vesicles followed by probe exchange between the vesicles in contact through SP-A. The contacts remain stable in the presence of calcium; i.e., the vesicles in contact do not change their partners on the time scale of several minutes. The binding of SP-A to vesicles and the aggregation of vesicles are rapid, and the aggregation is rapidly reversed by EGTA; i.e., both the forward and reverse aggregation reactions are complete in about 1 min. The exchange rate of the various probes between aggregated vesicles below 1 mM calcium in the presence of NaCl shows selectivity, i.e., a modest dependence on the net anionic charge on vesicles and for the headgroup of the probe. Exchange with lower selectivity is seen at >2 mM Ca in the absence of NaCl. SP-A binding to vesicles does not show an absolute specificity for the phospholipid structure, but the time course of the subsequent changes does. The results suggest that SP-A contacts between phospholipid interfaces could mediate the exchange of phospholipid species (trafficking and sorting) between lung surfactant pools in the hypophase and all accessible phospholipid interfaces of the alveolar space.

Calcium↗
PubMed1998

Cationic residues 53 and 56 control the anion-induced interfacial k*cat activation of pancreatic phospholipase A2.

Added NaCl or anionic amphiphiles increase the rate of hydrolysis of dispersions of zwitterionic phospholipid by pancreatic phospholipase A2 (PLA2). Two effects of the negative charge at the interface have been dissected: enhanced binding of the enzyme to the interface, and k*cat activation of the enzyme at the interface [Berg et al. (1997) Biochemistry 36, 14512-14530]. Results reported here show that the structural basis for the k*cat activation is predominantly through cationic K53 and K56 in bovine pancreatic PLA2 with the anionic interface. The maximum rate at saturating diheptanoylphosphatidylcholine micelles, VMapp, for WT, K56M, and K53M in 4 M NaCl is in the 800-1300 s-1 range. In contrast, VMapp at 0.1 M NaCl is considerably higher for K56M (400 s-1) and K53M (230 s-1) compared to the rate with WT (30 s-1) or K56E (45 s-1). The rate of hydrolysis of anionic dimyristoylphosphatidylmethanol vesicles is virtually the same with all these mutants (200-300 s-1) and it is not affected by added NaCl. The chemical step for the hydrolysis of anionic and zwitterionic substrates remains rate-limiting in the presence or absence of added NaCl. A modest (approximately 10-fold) effect of K56M substitution or of added NaCl is seen on the binding of the enzyme to the interface; however, the binding of the substrate or a substrate mimic to the active site of the enzyme at the interface is not affected by more than a factor of 2. Magnitudes of the primary rate and equilibrium parameters at the zwitterionic and anionic interfaces show that the effect of mutation or of added NaCl is primarily on k*cat at the zwitterionic interface. These results are interpreted in terms of a two-state model for the interfacial allosteric activation, where the enzyme-substrate complex at the zwitterionic interface becomes catalytically active only after the positive charge on cationic K56 and K53 has been removed by mutation or neutralized by anionic charges in the interface.

Amino Acid Substitution↗
PubMed1998

Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation.

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.

Adult↗
PubMed1998

Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B.

Cationic antimicrobial peptides, such as polymyxin B (PxB), below growth inhibitory concentration induce expression of osmY gene in viable E. coli without leakage of solutes and protons. osmY expression is also a locus of hyperosmotic stress response induced by common food preservatives, such as hypertonic NaCl or sucrose. High selectivity of PxB against Gram-negative organisms and the basis for the hyperosmotic stress response at sublethal PxB concentrations is attributed to PxB-induced mixing of anionic phospholipid between the outer layer of the cytoplasmic membrane with phospholipids in the inner layer of the outer membrane. This explanation is supported by PxB-mediated rapid and direct exchange of anionic phospholipid between vesicles. This mechanism is consistent with the observation that genetically stable resistance against PxB could not be induced by mutagenesis.

Anti-Bacterial Agents↗
PubMed1998

Interfacial recognition by bee venom phospholipase A2: insights into nonelectrostatic molecular determinants by charge reversal mutagenesis.

The basis for tight binding of bee venom phospholipase A2 (bvPLA2) to anionic versus zwitterionic phospholipid interfaces is explored by charge reversal mutagenesis of basic residues (lysines/arginines to glutamates) on the putative membrane binding surface. Single-site mutants and, surprisingly, multisite mutants (2-5 of the 6 basic residues mutated) are fully functional on anionic vesicles. Mutants bind tightly to anionic vesicles, and active-site substrate and Ca2+ binding are not impaired. Multisite mutants undergo intervesicle exchange slightly faster than wild type, especially in the presence of salt. It is estimated that electrostatic contribution to interfacial binding is modest, perhaps 2-3 kcal/mol of the estimated 15 kcal/mol. Elution properties of bvPLA2 from HPLC columns containing solid phases of tightly packed monolayers of phosphocholine amphiphiles suggest that ionic effects provide a modest portion of the interfacial binding energy and that this contribution decreases as the number of cationic residues mutated is increased. These results are consistent with the observation that Gila monster venom PLA2 (Pa2), which is homologous to bvPLA2, has high activity on anionic vesicles despite the fact that it has only a single basic residue on its putative interfacial recognition face. Results with bvPLA2 mutants show that manoalogue and 12-epi-scalaradial inactivate bvPLA2 by modification of K94. Also, deletion of the large beta-loop (residues 99-118) is without consequence for interfacial binding and catalysis of bvPLA2. All together, the preferential binding of bvPLA2 to anionic vesicles versus phosphatidylcholine vesicles is mainly due to factors other than electrostatics. Therefore hydrogen-bonding and hydrophobic interactions must provide a major portion of the interfacial binding energy, and this is consistent with recent spectroscopic studies.

Animals↗
PubMed1998

Interfacial activation of triglyceride lipase from Thermomyces (Humicola) lanuginosa: kinetic parameters and a basis for control of the lid.

A strategy is developed to analyze steady-state kinetics for the hydrolysis of a soluble substrate partitioned into the interface by an enzyme at the interface. The feasibility of this approach to obtain interfacial primary kinetic and equilibrium parameters is demonstrated for a triglyceride lipase. Analysis for phospholipase A2 catalyzed hydrolysis of rapidly exchanging micellar (Berg et al. (1997) Biochemistry 36, 14512-14530) and nonexchangeable vesicular (Berg et al., (1991) Biochemistry 30, 7283-7291) phospholipids is extended to include the case of a substrate that does not form the interface. The triglyceride lipase (tlTGL) from Thermomyces (formerly Humicola) lanuginosa hydrolyzes p-nitrophenylbutyrate or tributyrin partitioned in the interface of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) vesicles at a rate that is more than 100-fold higher than that for the monodispersed substrate or for the substrate partitioned into zwitterionic vesicles. Catalysis and activation is not seen with the S146A mutant without the catalytic serine-146; however, it binds to the POPG interface with the same affinity as the WT. Thus POPG acts as a diluent surface to which the lipase binds in an active, or "open", form for the catalytic turnover; however, the diluent molecules have poor affinity for the active site. Analysis of the substrate and the diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. As a competitive substrate, tributyrin provided a check for the apparent affinity parameters. Nonidealities from the fractional difference in the molecular areas in interfaces are expressed as the area correction factor and can be interpreted as a first-order approximation for the interfacial activity coefficient. The basis for the interfacial activation of tlTGL on anionic interface is attributed to cationic R81, R84, and K98 in the "hinge" around the 86-93 "lid" segment of tlTGL.

Amino Acid Sequence↗
PubMed1998

Thy-1, a novel marker for angiogenesis upregulated by inflammatory cytokines.

We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti-Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1-Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.

Angioplasty, Balloon↗
PubMed1998

Reductive dechlorination of DDE to DDMU in marine sediment microcosms.

DDT is reductively dechlorinated to DDD and dehydrochlorinated to DDE; it has been thought that DDE is not degraded further in the environment. Laboratory experiments with DDE-containing marine sediments showed that DDE is dechlorinated to DDMU in both methanogenic and sulfidogenic microcosms and that DDD is dehydrochlorinated to DDMU three orders of magnitude more slowly. Thus, DDD does not appear to be an important precursor of the DDMU found in these sediments. These results imply that remediation decisions and risk assessments based on the recalcitrance of DDE in marine and estuarine sediments should be reevaluated.

Anaerobiosis↗
PubMed1998

Molecular cloning, characterization, and promoter analysis of the mouse Crp2/SmLim gene. Preferential expression of its promoter in the vascular smooth muscle cells of transgenic mice.

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5'-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5' of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5' of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5'-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5'-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases -74 and -39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.

Amino Acid Sequence↗
PubMed1998

Enhancement of serum-response factor-dependent transcription and DNA binding by the architectural transcription factor HMG-I(Y).

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Animals↗
PubMed1998

In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells.

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.

Animals↗
PubMed1998

Human EZF, a Krüppel-like zinc finger protein, is expressed in vascular endothelial cells and contains transcriptional activation and repression domains.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Amino Acid Sequence↗