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Biomedical subjects

V Gopalakrishnan

Publications and source records attributed to V Gopalakrishnan.

At least 55 records · Page 3Linked to original sources
PubMed1998

Insulin increases endothelin-1-evoked intracellular free calcium responses by increased ET(A) receptor expression in rat aortic smooth muscle cells.

While insulin is known to promote vascular smooth muscle (VSM) relaxation, it also enhances endothelin-1 (ET-1) secretion and action in conditions such as NIDDM and hypertension. We examined the effect of insulin pretreatment on intracellular free calcium ([Ca2+]i) responses to ET-1 in cultured aortic smooth muscle cells (ASMCs) isolated from Sprague-Dawley (SD) rats and measured ET(A) receptor characteristics and ET-1-evoked tension responses in aorta obtained from insulin-resistant, hyperinsulinemic Zucker-obese (ZO) and control Zucker-lean (ZL) rats. Pretreatment of rat ASMCs with insulin (10 nmol/l for 24 h) failed to affect basal [Ca2+]i levels but led to a significant increase in peak [Ca2+]i response (1.7-fold; P < 0.01) to ET-1. The responses to IRL-1620 (an ET(B) selective agonist), ANG II, and vasopressin remained unaffected. ET-1-evoked peak [Ca2+]i responses were significantly attenuated by the inclusion of the ET(A) antagonist, BQ123, in both groups. The ET(B) antagonist, BQ788, abolished [Ca2+]i responses to IRL-1620 but failed to affect the exaggerated [Ca2+]i responses to ET-1. Saturation binding studies revealed a twofold increase (P < 0.01) in maximal number of binding sites labeled by 125I-labeled ET-1 in insulin-pretreated cells and no significant differences in sites labeled by 125I-labeled IRL-1620 between control and treatment groups. Northern blot analysis revealed an increase in ET(A) mRNA levels after insulin pretreatment for 20 h, an effect that was blocked by genistein, actinomycin D, and cycloheximide. Maximal tension development to ET-1 was significantly greater (P < 0.01), and microsomal binding studies using [3H]BQ-123 revealed a twofold higher number of ET(A) specific binding sites (P < 0.01) in aorta from ZO rats compared with that of ZL rats. These data suggest that insulin exaggerates ET-1-evoked peak [Ca2+]i responses via increased vascular ET(A) receptor expression, which may contribute to enhanced vasoconstriction observed in hyperinsulinemic states.

Animals↗
PubMed1998

Enhanced detection of herpes simplex virus from ocular specimens of herpetic keratitis patients.

Corneal scrapings collected from 70 patients were used to assess the diagnostic value of indirect immunofluorescence (indirect IF) procedure in comparison with routine virus culture (RVC) for the diagnosis of Herpes simplex virus induced keratitis (HSK). Virus specific antigen was detected by indirect IF in 22 (31.42%) cases. In contrast, only 20% (14) of the cases had positive viral isolation which sometimes took as long as a week to show a cytopathogenic effect (CPE). It is concluded that antigen detection by indirect IF is a rapid, specific and sensitive technique for demonstrating HSV-1 antigen in corneal scrapings from HSK patients and a useful laboratory tool not only for diagnosing HSK but also for monitoring efficiency of anti HSV treatment for HSK.

Animals↗
PubMed1997

Endothelin contributes to the hemodynamic effects of vasopressin in spontaneous hypertension.

Changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of [Arg8]-vasopressin (vasopressin) were recorded before and after pretreatment with bosentan, a non-selective endothelin antagonist, in conscious unrestrained spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The presser effects of vasopressin were exaggerated in SHR compared to WKY. Pretreatment with bosentan failed to change hemodynamic responses of WKY to vasopressin, but it blunted the increases in blood pressure and the decreases in conductance evoked by vasopressin in SHR. In contrast, bosentan failed to change cardiac output responses of SHR to vasopressin. Except at the highest dose of vasopressin, bosentan abolished the exaggerated pressor responsiveness of the SHR to vasopressin. The results suggest that endothelin contributes to the exaggerated pressor responsiveness of SHR to vasopressin, and that this effect is exerted at the level of the resistance vessels and not on factors that regulate cardiac output.

Animals↗
PubMed1996

High-performance liquid chromatographic assay for the quantitation of L-arginine in human plasma.

L-Arginine is metabolized to nitric oxide by nitric oxide synthase, and abnormalities in nitric oxide production have been implicated in the pathogenesis of some diseases involving the vasculature. Thus, there has been interest in the effects of pharmacologic doses of L-arginine in patients with cardiovascular and renal diseases. To study the disposition of exogenous doses, an HPLC method was developed to analyze plasma samples for L-arginine. The assay involves precolumn derivatization of arginine with naphthalenedicarboxaldehyde and cyanide followed by HPLC with UV detection. Only a simple deproteinization of the plasma samples was required. The derivatized arginine was stable (less than 5% degradation in 20 h), facilitating batch sample processing and analysis in an autosampler. Calibration curves were generated in Ringer's lactate solution instead of plasma to correct for endogenous plasma L-arginine. Recovery in plasma, compared to Ringer's solution (n = 4), was 103%. Mean intraday assay precision (n = 6), expressed as coefficient of variation, was 3.4%. Interassay precision (n = 6) was 7%. The assay was applied for the quantitation of L-arginine in plasma samples from a normal subject who had been given a single oral (10 g) and a single intravenous dose (30 g) of exogenous L-arginine.

Arginine↗
PubMed1996

Actions of neurotoxic beta-amyloid on calcium homeostasis and viability of PC12 cells are blocked by antioxidants but not by calcium channel antagonists.

The fragment of beta-amyloid comprised of amino acids 25-35 induces a rapid, concentration-dependent increase in cytosolic free calcium levels in suspensions of PC12 neuronal cells. This action of beta-amyloid 25-35 is not altered by pretreatment with the calcium channel blockers nifedipine or cobalt, with the depleter of intracellular calcium stores cyclopiazonic acid, or with the phospholipase C inhibitor neomycin. However, the effects of beta-amyloid 25-35 on cytosolic free calcium are absent in calcium-free buffer and are blocked by the antioxidant lazaroid U-83836E and by vitamin E. beta-Amyloid 25-35 is also neurotoxic and produces a concentration-dependent reduction in the viability of PC12 cells in culture. The neurotoxic action of beta-amyloid is blocked by U-83836E and vitamin E but not by nifedipine or cobalt. These data indicate that both the disruption of calcium homeostasis and the reduction of cell viability produced by beta-amyloid in PC12 cells are mediated by free radical-based processes.

Amyloid beta-Peptides↗
PubMed1996

Effect of an endothelin antagonist on hemodynamic responses to angiotensin II.

We determined changes in blood pressure, cardiac output, and total peripheral conductance evoked by intravenous infusions of angiotensin II (Ang II) in conscious, unrestrained normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after pretreatment with bosentan, a nonselective endothelin antagonist. Blood pressure was recorded by radiotelemetry and cardiac output by ultrasonic transit-time flow probes. Bosentan per se failed to affect basal blood pressure and evoked only small changes in cardiac output and total peripheral conductance in both strains. The pressor effects of Ang II were exaggerated in SHR compared with WKY. Strikingly, bosentan pretreatment blunted the increases in blood pressure, the fall in cardiac output, and the decreases in conductance evoked by lower doses of Ang II but not higher doses of the peptide. This effect was observed in both rat strains but was more pronounced in SHR. These data suggest that endothelin contributes to the hemodynamic effects of Ang II in both SHR and WKY and that endothelin may contribute to the exaggerated pressor responsiveness of SHR to Ang II.

Angiotensin II↗
PubMed1995

Stimulation of human papillomavirus type 1a DNA replication by a multimerized AT-rich palindromic sequence.

Replication of most papillomaviruses requires the viral E1 and E2 proteins and an origin of replication containing the E1 and E2 binding sites. In the case of human papillomavirus type 1a (HPV-1a), the E1 protein alone is sufficient for DNA replication although the E2 protein significantly stimulates replication. We have further analyzed the role of cis-acting sequences and the E1 and E2 proteins in HPV-1a replication. Previous studies have shown that a 60-bp region lacking the E2 binding sites but containing an imperfect 16-bp AT-rich palindrome corresponding to the putative E1 binding site contains the minimal origin of replication (ori). Using a transient replication assay, we demonstrate that duplication of this 60-bp region causes a severalfold increase in replication. Synthetic oligonucleotides containing a 39-bp region centered around the above palindromic sequence supported only low-level replication in the presence of either E1 alone or both E1 and E2, but replication was significantly increased in the presence of multiple copies of this sequence. Plasmids containing a 19-bp sequence which includes the AT-rich palindrome failed to replicate, but multiple copies of this region supported replication in the presence of both E1 and E2 to significant levels. The results presented indicate that the HPV-1a E1 protein is capable of recruiting all the cellular factors required for replication. Our results also suggest that multimerization of the AT-rich palindromic sequence may result in a significant increase in the recruitment of the E1 protein to the origin, thereby stimulating replication. This increased targeting of the E1 protein to the origin containing multiple copies of the putative E1 binding site may be functionally similar to the targeting of E1 to the origin by E2.

Adenine↗
PubMed1995

Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization.

A fundamental catalytic principle for protein enzymes in the use of binding interactions away from the site of chemical transformation for catalysis. We have compared the binding and reactivity of a series of oligonucleotide substrates and products of the Tetrahymena ribozyme, which catalyzes a site-specific phosphodiester cleavage reaction: CCCUCUpA+G<-->CCCUCU-OH+GpA. The results suggest that this RNA enzyme, like protein enzymes, can utilize binding interactions to achieve substantial catalysis via entropic fixation and substrate destabilization. The stronger binding of the all-ribose oligonucleotide product compared to an analog with a terminal 3' deoxyribose residue gives an effective concentration of 2200 M for the 3' hydroxyl group, a value approaching those obtained with protein enzymes and suggesting the presence of a structurally well defined active site capable of precise positioning. The stabilization from tertiary binding interactions is 40-fold less for the oligonucleotide substrate than the oligonucleotide product, despite the presence of the reactive phosphoryl group in the substrate. This destabilization is accounted for by a model in which tertiary interactions away from the site of bond cleavage position the electron-deficient 3' bridging phosphoryl oxygen of the oligonucleotide substrate next to an electropositive Mg ion. As the phosphodiester bond breaks and this 3' oxygen atom develops a negative charge in the transition state, the weak interaction of the substrate with Mg2+ becomes strong. These strategies of "substrate destabilization" and "transition state stabilization" provide estimated rate enhancements of approximately 280- and approximately 60-fold, respectively. Analogous substrate destabilization by a metal ion or hydrogen bond donor may be used more generally by RNA and protein enzymes catalyzing reactions of phosphate esters.

Animals↗
PubMed1995

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells.

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca2+]i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10-200 microM) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentration-dependent increase in [Ca2+]i with an EC50 (mean +/- SEM) value of 42 +/- 11 microM. Inclusion of vanadate (200 microM) led to a significant increase (p < 0.05) in the peak [Ca2+]i level to 190 +/- 23 nM from a basal level of 102 +/- 2 nM. At concentrations > 200 microM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50% (due to a quenching effect), followed by a transient rise. H2O2, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca2+]i. These data suggest that vanadate promotes vascular tone by elevating [Ca2+]i in ASMC. However, [Ca2+]i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.

Animals↗
PubMed1995

Differential effects of phosphoramidon on contractile responses to angiotensin II in rat blood vessels.

1. Cumulative concentration-tension response (C-R) curves to angiotensin II (AII), big endothelin-1 (big ET-1), ET-1 and arginine vasopressin (AVP) were determined in endothelium intact-ring preparations of aorta, mesenteric artery and tail artery isolated from adult male Sprague-Dawley rats in the presence or absence of the neutral metalloprotease inhibitor, phosphoramidon. 2. The order of sensitivity of the three rat vascular smooth muscle preparations to AII, big ET-1 and ET-1 was aorta > mesenteric artery > tail artery whereas that for AVP was reversed, namely, tail artery > mesenteric artery > aorta. 3. Phosphoramidon blocked the responses to AII in a concentration-dependent manner, whereas even very high concentrations of phosphoramidon (100 microM) failed to affect the tension responses evoked by ET-1 and AVP in all three preparations. Low concentrations of phosphoramidon (10 microM) produced significant increases in EC50 values for AII in tail artery (P < 0.01) and mesenteric artery (P < 0.05) but not in aorta. The rank order of sensitivity to the inhibition by phosphoramidon was tail artery > mesenteric artery > aorta. Phosphoramidon-evoked rightward shifts in the C-R curves to AII were much higher than those to big ET-1 in both mesenteric artery and tail artery. 4. In endothelium-denuded preparations, AII failed to evoke any increases in tension in tail artery while the responsiveness of the mesenteric artery to AII was reduced significantly relative to endothelium-intact tissues with a rightward shift in the C-R curve and a decrease in the maximal response. On the other hand, the C-R curve to AII was shifted to the left in aorta following removal of the endothelium.Importantly, ET-1 and AVP evoked vasoconstrictor responses were unaffected by the inclusion of a high concentration of phosphoramidon (100 microM) in endothelium-denuded aorta and mesenteric artery.5. The results suggest that AII-evoked tension responses of blood vessels such as tail artery are completely endothelium-dependent; in relatively larger blood vessels such as mesenteric artery they are partially endothelium-dependent while in much bigger conduit type blood vessel such as aorta, they are endothelium-independent. It is concluded that the vasoconstrictor responses to AII in mesenteric artery and tail artery may be mediated by the release of endothelins from the endothelium by increased formation from big ET, an effect that is blocked by phosphoramidon.

Angiotensin II↗
PubMed1995

Heterogeneity in vascular smooth muscle responsiveness to angiotensin II. Role of endothelin.

We compared the role of endothelium and of endothelin in mediating the vasoconstrictor responses to angiotensin II (Ang II) in three vascular smooth muscle preparations--aorta, mesenteric artery, and tail artery--isolated from adult male Sprague-Dawley rats. The vasoconstrictor potency for Ang II in blood vessels with endothelium varied in the following rank order: aorta > mesenteric artery > tail artery. Although the maximal tension responses to Ang II were similar for mesenteric and tail arteries, it was significantly lower in aorta. Endothelium removal led to a leftward shift in the concentration-response curves to Ang II in the aorta but a rightward shift in the mesenteric artery. Strikingly, Ang II failed to evoke tension responses in tail artery in the absence of endothelium. The endothelin-A (ETA)-selective antagonist BQ-123 blocked the responses to Ang II in a noncompetitive manner, with partial and complete attenuation of responses in the endothelium-intact mesenteric and tail artery preparations, respectively. In contrast, BQ-123 did not affect the responses to Ang II in the aorta. BQ-123 also failed to affect the responses to Ang II in endothelium-denuded mesenteric artery rings. The Ang II type 1 (AT1) receptor-selective antagonist losartan competitively blocked the responses to Ang II in the three tissues (pA2, 8.3 to 8.7) when endothelium was present. These data suggest that there are endothelium-dependent regional variations in vascular tissue sensitivity to Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1994

E1 protein of human papillomavirus type 1a is sufficient for initiation of viral DNA replication.

Previous studies on transient replication of papillomaviruses have shown an absolute requirement for the viral E1 and E2 proteins in DNA replication. Here we demonstrate that for human papillomavirus type 1a (HPV-1a) DNA, the E1 protein alone is sufficient for in vivo replication of plasmids containing the viral origin of replication. Replication was origin-specific and required the presence of a DNA sequence containing a putative E1 binding site, but the E2 binding sites were dispensable. In the presence of the E1 protein, E2 stimulated replication of plasmids containing the E1 and E2 binding sites, but no stimulation was observed when the origin plasmids lacked E2 binding sites. Conversely, in the presence of E1 alone, the E2 binding sites did not affect replication. Plasmids containing the replication origins of HPV-6b, HPV-18, and bovine papillomavirus type 1 (BPV-1) also replicated efficiently in the presence of the HPV-1a E1 and E2 proteins. However, plasmids containing the origins of HPV-6b and HPV-18 failed to replicate in the presence of HPV-1a E1 alone, whereas a plasmid containing the BPV-1 origin replicated to lower levels than the HPV-1a origin-containing plasmid. These results suggest that replication from papillomaviral origins in the presence of E1 alone is presumably dependent on the strength of E1-origin interactions. Additionally, E1-dependent replication is stimulated by the E2 protein in the presence of E2 binding sites.

Base Sequence↗
PubMed1994

Spatial relationship between polymerase and exonuclease active sites of phage T4 DNA polymerase enzyme.

The spatial relationship between the polymerase and exonuclease active sites of bacteriophage T4 DNA polymerase enzyme has been examined using a bulky biotin-streptavidin block at a specified position in an oligonucleotide (Fig. 1). The idea was to monitor the closest distance of approach of the T4 enzyme before being blocked by the bulky biotin-streptavidin complex while performing either of its activities. The results indicated a distance of 4-5 nucleotides between the biotin-streptavidin probe and the exonuclease site and a distance requirement of at least 7 nucleotides between the bulky probe and the 3'-primer terminus for efficient polymerization by the T4 enzyme. The difference in the two distances suggested a separation of 2-3 nucleotides between the two active sites of the T4 enzyme.

Bacterial Proteins↗
PubMed1994

Effect of a thiobenzimidazolone derivative on DNA strand transfer catalyzed by HIV-1 reverse transcriptase.

Thiobenzimidazolone (TIBO) derivatives are known inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The effect of a TIBO derivative ((+)-S-4,5,6,7-tetrahydro-9-chloro-5- methyl-6-(3-methyl-2-butenyl)-imidazol[4,5,1-jk]1,4-benzodiazapine -2-thione ) on the DNA strand transfer reaction catalyzed by HIV-1 RT (which is a function of both the DNA polymerase and RNase H activities) was investigated by delineating the effect of the drug on the constitutive DNA polymerase and RNase H activities) was investigated by delineating the effect of the drug on the constitutive DNA polymerase and RNase H activities. Single nucleotide incorporation on template-primer 1 was used to study the DNA polymerase activity of HIV-1 RT while template-primer 2 was used to study the effect of TIBO on the RNase H activity (polymerase independent). The drug was found to decrease the amplitude of the presteady-state burst when preequilibrated with the enzyme-substrate complex besides decreasing the steady-state rate of single nucleotide incorporations. In the absence of preincubation, TIBO did not affect the burst amplitude but decreased the steady-state rate after the pre-transient phase. This suggested that binding of TIBO to RT was affected by the presence of template-primer and required dissociation of the enzyme from the template-primer for effective binding. The polymerase-independent RNase H activity was activated in the presence of TIBO. The effect of TIBO on the overall process of DNA strand transfer is a balance between its inhibition of the polymerase activity and its activation of the RNase H activity.

Antiviral Agents↗
PubMed1994

Angiotensin II elevates cytosolic free calcium in human lung adenocarcinoma cells via activation of AT1 receptors.

Angiotensin II (Ang II), bradykinin (BK), and endothelin-1 (ET-1) evoked alterations in cytosolic free calcium, [Ca2+]i, levels were determined using fura-2 fluorescence methodology in a human lung adenocarcinoma cell line (A549), a non-neoplastic lung cell line and a small cell lung carcinoma cell (SCLC) line. Ang II and BK evoked a rapid, concentration-dependent transient increase in [Ca2+]i in A549 cells. The peak [Ca2+]i increases attained with Ang II (1 microM) and BK (1 microM) were 3- and 4-fold higher, respectively (P < 0.01) than the basal [Ca2+]i values. This effect of Ang II was completely abolished by inclusion of losartan (DuP 753), an AT1 subtype selective antagonist. Removal of extracellular Ca2+ from the incubation medium led to significant diminution of the peak [Ca2+]i response to Ang II but not to BK. In contrast to Ang II and BK, ET-1 failed to evoke an increase in [Ca2+]i levels in A549 cells. Neither Ang II nor ET-1 evoked any appreciable increase in [Ca2+]i levels of non-neoplastic lung cell and SCLC cell lines. These data confirm that the human non-small cell lung cancer cells (A549) selectively express AT1 subtype receptors for Ang II that are functionally coupled to Ca2+ mobilization from both extra and intracellular sources.

Adenocarcinoma↗
PubMed1994

High glucose attenuates peptide agonist-evoked increases in cytosolic free [Ca2+] in rat aortic smooth muscle cells.

Incubation of cultured rat aortic smooth muscle cells (ASMCs) in a medium containing high glucose concentrations (25 mM) did not affect the basal cytosolic free calcium ([Ca2+]i) but led to significant reductions in peak [Ca2+]i response evoked by arginine vasopressin, angiotensin II, and endothelin-1 (ET-1). This was observed in both the presence and absence of extracellular Ca2+. Maintenance of rat ASMCs in a medium containing mannose (an osmotic control for high glucose) did not affect either the basal or peptide agonist-evoked increase in [Ca2+]i. However, pretreatment with either the nonselective protein kinase C (PKC) inhibitor staurosporine or the selective PKC inhibitor 2,6-diamino-N-([1-(1-oxotridecyl)-2 piperidinyl] methyl) hexanamide reversed the attenuating effect of high glucose on peak [Ca2+]i response evoked by ET-1. Also, short-term incubation of ASMCs with the active phorbol ester, phorbol 12-myristate 13-acetate, led to a reduction in peak [Ca2+]i response to all three agonists, whereas the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, had no such effect. Although high-glucose treatment of rat ASMCs led to significant reductions in the maximal number of binding sites to the extent of 39% of [125I]ET-1 specific binding, no significant differences in the affinity (Kd approximately 110 pM) characteristics were evident between control and high-glucose treatment groups. It is proposed that incubation of rat ASMCs with high glucose enhances the de novo synthesis of diacylglycerol and activates membrane-bound PKC and that this, in turn, impairs agonist-mediated intracellular Ca2+ mobilization.

Alkaloids↗
PubMed1994

Induction of rules for biological macromolecule crystallization.

X-ray crystallography is the method of choice for determining the 3-D structure of large macromolecules at a high enough resolution. The rate limiting step in structure determination is the crystallization itself. It takes anywhere between a few weeks to several years to obtain macromolecular crystals that yield good diffraction patterns. The theory of forces that promote and maintain crystal growth is preliminary, and crystallographers systematically search a large parameter space of experimental settings to grow good crystals. There is a wealth of experimental data on crystal growth most of which is in paper laboratory notebooks. Some of the data has been gathered in electronic form, e.g., the Biological Macromolecular Crystallization Database (BMCD) which is a repository of successful experimental conditions for growing over 800 different macromolecules (Gilliland 1987). Crystallographers are in need of computational tools to gather and analyze past data to design new crystal growth trails. We are building the Crystallographer's Assistant (CA) to help crystallographers record and maintain experimental context in electronic form, offer suggestions on experimental conditions that are likely to be successful, and provide explanations for failed experiments. As an initial step in this project, we have applied RL, an inductive learning program, to the BMCD. In this paper we report initial experiments and findings in applying RL to the BMCD. From the point of view of crystallography, we have discovered possibly significant new empirical relationships in crystal growth. From the point of view of machine learning, our work suggests refinements of existing methods for incorporating detailed domain knowledge into inductive analysis techniques.

Animals↗