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K Venkateswarlu

Publications and source records attributed to K Venkateswarlu.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular cloning and functional characterization of a human homologue of centaurin-alpha.

We report here the molecular cloning, expression, and characterisation of a human homologue of rat centaurin-alpha, which we have termed centaurin-alpha(1). The cDNA contains a single open reading frame, which encodes a 373-amino-acid protein with a calculated molecular weight of 43,429 Daltons. Centaurin-alpha(1) shows high identity at the amino acid level with the other centaurin-alpha homologues, p42(IP4) and PIP(3)BP. Northern analysis revealed that centaurin-alpha(1) expresses as a single 2.5-kb transcript, mainly in the brain. Recombinant centaurin-alpha(1) binds the inositol head group of PtdIns(3,4,5)P(3) and Ins(1,3,4, 5)P(4), with high affinity (K(d) 139.7 +/- 10.5 nM) and inositol phosphate specificity, consistent with it functioning as a putative PtdIns(3,4,5)P(3) receptor. In keeping with this conclusion, we have shown that GFP-tagged centaurin-alpha(1) recruits to the plasma membrane in a PI 3-kinase-dependent manner and the recruitment is inhibited by the PI 3-kinase inhibitor wortmannin. These results suggest that centaurin-alpha(1) can function as an in vivo PtdIns(3, 4,5)P(3) receptor.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Generation of a complete, soluble, and catalytically active sterol 14 alpha-demethylase-reductase complex.

Sterol 14 alpha-demethylation is one of the key steps of sterol biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-demethylase (other names being CYP51 and P45014DM) encoded by ERG11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by NCPR1 (NCP1), which is also associated with the endoplasmic reticulum. A diglycine linker recognition site (Gly-Gly-Ile-Glu-Gly-Arg-Gly-Gly) for the protease factor Xa, also containing a thrombin recognition site, was inserted just beyond the N-terminal hydrophobic segment of Candida albicans Erg11p. This modified enzyme was heterologously expressed at a level of 2.5 nmol of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Following purification, treatment of the modified protein with factor Xa or thrombin resulted in sequence-specific cleavage and production of a soluble N-terminal truncated Erg11p which exhibited spectral characteristics identical to those of the purified full-length, wild-type form. Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system. The complex was disrupted by high-salt concentration, reflecting the importance of electrostatic forces in the protein-protein interaction. The results demonstrate the membrane anchor serves to localize Erg11p to the ER where the substrate is located, but is not essential in either Ncpr1p or Erg11p activity. The possibility of cocrystallization of an active soluble eukaryotic 14 alpha-demethylase can be envisaged.

Amino Acid Sequence↗
PubMed1999

Identification of centaurin-alpha1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

ADP-Ribosylation Factors↗
PubMed1999

Identification of the ras GTPase-activating protein GAP1(m) as a phosphatidylinositol-3,4,5-trisphosphate-binding protein in vivo.

GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs) [1]. In vitro, it has been shown to bind inositol 1, 3,4,5-tetrakisphosphate (IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate (PIP3) [2] [3]. This has led to the suggestion that GAP1(m) might function as a PIP3 receptor in vivo [4]. Here, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a chimera of green fluorescent protein fused to GAP1(m) (GFP-GAP1(m)), we show that epidermal growth factor (EGF) induces a rapid (less than 60 seconds) recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment required a functional GAP1(m) pleckstrin homology (PH) domain, because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro [5] totally blocked EGF-induced GAP1(m) translocation. Furthermore, the membrane translocation was dependent on PI 3-kinase, and the time course of translocation paralleled the rate by which EGF stimulates the generation of plasma membrane PIP3 [6]. Significantly, the PIP3-induced recruitment of GAP1(m) did not appear to result in any detectable enhancement in its basal Ras GAP activity. From these results, we conclude that GAP1(m) binds PIP3 in vivo, and it is recruited to the plasma membrane, but does not appear to be activated, following agonist stimulation of PI 3-kinase.

Animals↗
PubMed1999

Effects of heptanol on the neurogenic and myogenic contractions of the guinea-pig vas deferens.

1. The effects of the putative gap junction uncoupler, 1-heptanol, on the neurogenic and myogenic contractile responses of guinea-pig vas deferens were studied in vitro. 2. Superfusion of 2.0 mM heptanol for 20-30 min produced the following reversible changes in the biphasic neurogenic contractile response (8 trials): (i) suppression of both phases; (ii) delayed development of both the first as well as the second phase, accompanied by complete temporal separation of the two phases; (iii) prominent oscillations of force during the second (noradrenergic) phase only. 3. To eliminate prejunctional effects of heptanol, myogenic contractions were evoked by field stimulation of the vas in the presence of suramin (200 microM) and prazosin (1 microM). Heptanol (2.0 mM) abolished these contractions reversibly. 4. These results show that (i) heptanol inhibits both excitatory junction potential (EJP)-dependent and non EJP-dependent contractions of the vas; (ii) a postjunctional site of action of heptanol, probably intercellular uncoupling of smooth muscle cells, contributes to the inhibition of contraction.

Adenosine Triphosphate↗
PubMed1999

Potential regulation of ADP-ribosylation factor 6 signalling by phosphatidylinositol 3,4,5-trisphosphate.

In this review we have described data supporting the conclusion that PtdIns(3,4,5)P3 may regulate the activation state of ARF6 through an ability to recruit the ARF exchange factors ARNO, GRP1 and cytohesin-1 to the plasma membrane. The downstream consequences of such a PtdIns(3,4,5)P3-dependent activation of ARF6 are presently unclear. However, given the role of ARF6 in fusion events at the plasma membrane, we have proposed that PtdIns(3,4,5)P3 may regulate vesicle trafficking at this membrane through its ability to activate ARF6. This is an attractive possibility given the number of PtdIns(3,4,5)P3-dependent pathways which involve some aspect of vesicle trafficking at the plasma membrane, for instance glucose transport, membrane ruffling and cell movement.

ADP-Ribosylation Factor 6↗
PubMed1999

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

Animals↗
PubMed1999

Ion Channels and Gap Junctions: Their Role in Erectile Physiology, Dysfunction, and Future Therapy.

A flurry of research and clinical activity during this past decade has documented that the tonicity and synchronicity of the corporal smooth muscle cells of the penis are major determinants of erectile capacity and function. Specifically, the effects of diverse and bifurcating intracellular signal transduction pathways on the activity of nonjunctional ion channels such as potassium (K(+)), calcium (Ca(2+)), and chloride (C(1-)) govern the former, whereas intercellular communication through gap junctions provides the anatomic substrate for the latter. Recent studies at the tissue, cellular, subcellular, and molecular levels have verified this supposition and provided important insight into how subtle alterations in the balance between contraction and relaxation of the corporal smooth muscle cells can predispose a man to erectile failure. This report reviews the available information concerning the participation of gap junctions and K(+), Ca(2+), and C(1-) channels in the erectile process and describes their importance as potential molecular targets for the future therapy of erectile dysfunction (ED). It is argued that a major goal should now be to proceed on at least two fronts simultaneously: (1) to capitalize on these new mechanistic insights by developing novel treatments for ED centered on the modulation of ion channel activity; and (2) simultaneously to take advantage of the unique therapeutic opportunities afforded by the presence and ubiquitous distribution of gap junction channels in the human corpora. One strategy that fulfils both criteria will be briefly reviewed, that is, gene therapy with the maxi-K(+) channel subtype.

Journal Article↗
PubMed1998

Nerve growth factor- and epidermal growth factor-stimulated translocation of the ADP-ribosylation factor-exchange factor GRP1 to the plasma membrane of PC12 cells requires activation of phosphatidylinositol 3-kinase and the GRP1 pleckstrin homology domain.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

ADP-Ribosylation Factors↗
PubMed1998

Estimation of variance components based on diallel model.

The problem of estimation of variance components based on diallel model for unbalanced data has been addressed. The least squares approach to quadratic estimation has been adopted in obtaining the explicit solutions for the design and genetic components of variance.

Alleles↗
PubMed1998

Insulin-dependent translocation of ARNO to the plasma membrane of adipocytes requires phosphatidylinositol 3-kinase.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells [1]. ARNO is a member of the family of guanine nucleotide exchange factors for ARFs which includes cytohesin-1 and GRP-1 [2] [3-5]. Members of this family contain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) in vitro [3,4]. Here, we show that recombinant ARNO has the binding characteristics of a PIP3 receptor and that this activity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is localised exclusively in the cytoplasm. Stimulation with insulin, however, causes a rapid (< 50 second) PH-domain-dependent translocation of GFP-ARNO to the plasma membrane. This translocation is blocked by the PI(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002, and by co-expression with a dominant-negative p85 mutant, suggesting that the translocation is a consequence of insulin stimulation of PI 3-kinase. Our data strongly suggest that ARNO binds PIP3 in vivo and that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.

3T3 Cells↗
PubMed1998

The N-terminal membrane domain of yeast NADPH-cytochrome P450 (CYP) oxidoreductase is not required for catalytic activity in sterol biosynthesis or in reconstitution of CYP activity.

The disruption of Saccharomyces cerevisiae NADPH- cytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.

Catalysis↗
PubMed1998

NADPH cytochrome P-450 oxidoreductase and susceptibility to ketoconazole.

The phenotype of a strain of Saccharomyces cerevisiae containing a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hypersensitivity to the alternative electron donor system in cpr strains. Both phenotypes could be reversed in transformants expressing the CPR and FUS, indicating the availability of the CPR in FUS as well as the expressed native CPR for monoxygenase-associated reactions. The complementation of function was observed both in vitro and in vivo for the monoxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway with which CPR is coupled. Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents.

Antifungal Agents↗
PubMed1997

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1IP4BP and GAP1m result from the GAP1IP4BP PH domain directing plasma membrane targeting.

Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.

Animals↗
PubMed1997

Effects of heptanol on electrical activity in the guinea-pig vas deferens.

1. The effects of the putative intercellular uncoupling agent I-heptanol on electrical activity in the guinea-pig vas deferens were studied by use of intracellular and extracellular recording techniques. 2. At concentrations of 0.5, 1 and 2 mM, heptanol rapidly, monotonically and reversibly attenuated intracellularly recorded excitatory junction potential (e.j.p.) amplitude without affecting its time course, while spontaneous excitatory junction potentials (s.e.j.ps) were left unaffected. 3. Heptanol did not affect either the extracellularly recorded evoked excitatory junction current (e.j.c.), or the nerve terminal impulse that preceded it. These observations indicate that heptanol does not affect nerve impulse conduction, neurotransmitter release, or the postjunctional receptors involved in the production of the e.j.p. 4. E.j.ps appear to be suppressed by heptanol due to its intercellular uncoupling effects. Therefore, functional intercellular coupling may be necessary for the generation of the e.j.p. in smooth muscle.

Alcohols↗
PubMed1997

Stereoselective interaction of SCH 39304, a triazole, with sterol 14alpha-demethylase of Aspergillus fumigatus.

The inhibitory activity of SCH 39304 and its enantiomers on radial growth and on the target enzyme, sterol 14alpha-demethylase, in Aspergillus fumigatus was studied to assess the role of stereochemistry in the efficacy of the drug. SCH 39304 and the RR(+) enantiomer were active in inhibiting the growth while no inhibition in the growth was observed with the SS(-) enantiomer. The MIC of SCH 39304 for the growth was about twice that of the RR(+) enantiomer. The differences in IC50s of SCH 39304 and its enantiomers for cell-free ergosterol biosynthesis correlated with their variations in MICs and type II binding spectra indicated the SS(-) enantiomer failed to bind to microsomal P450. These results show that the difference between SS(-) and RR(+) enantiomers in interacting with the target enzyme is the cause for significant difference in the potency between these two forms.

Antifungal Agents↗