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S Vasudevan

Publications and source records attributed to S Vasudevan.

At least 19 recordsLinked to original sources
PubMed1995

Characterization of the rat m3 muscarinic acetylcholine receptor produced in insect cells infected with recombinant baculovirus.

The m3 muscarinic acetylcholine receptor from rat heterologously produced in insect cells after infection with a recombinant baculovirus has an apparent molecular mass of approximately 75 kDa. Polyclonal antibodies raised against a carboxy-terminal nonapeptide that is unique to the m3 subtype can detect the receptors produced in the insect cells by Western blot and can also immunoprecipitate solubilized receptor. Immunofluorescence microscopy as well as electron microscopy revealed that the receptor was located intracellularly, visualized as a ring around the nucleus of the infected insect cells. Solubilization of the receptor was accomplished with digitonin which was added in increments (over 10 min) to a final concentration of 0.8% (mass/vol). The solubilized receptor is unstable when the ligand-binding site is not protected by a ligand. Here the low-affinity ligand propylbenzilylcholine (approximately 10 nM) has demonstrable protective ability during solubilization, but the usefulness of this ligand is limited by a very slow off rate. From the behaviour of the solubilized receptor during DEAE-Sephacel chromatography and lectin-affinity chromatography it can be deduced that the receptor produced in insect cells is heterogeneously glycosylated in the producing insect cells.

Affinity Labels↗
PubMed1995

Nucleotide pool imbalances in the livers of patients with urea cycle disorders associated with increased levels of orotic aciduria.

Liver samples obtained at autopsy from patients with ornithine transcarbamylase (OTC) deficiency, a urea cycle disorder that is associated with high levels of orotic acid biosynthesis and excretion were analysed for nucleotide pools. As a control, liver samples from patients with a deficiency of mitochondrial carbamyl phosphate synthetase (CPS-I) which is not associated with increased levels of orotic acidurias were also analysed. The results show that liver tissue from OTC deficiency patients exhibited an increased ratio of uridine nucleotides to adenosine nucleotides, while in CPS-I deficiency patients, no such increase was noted. This study indicates that genetic disorders that are associated with increased loads of orotic acid exhibit abnormally high ratios of uridine to adenosine nucleotides in the liver. This type of imbalance is analogous to that seen in the liver of rats and mice exposed to an orotic acid supplemented or an arginine-deficient diet under liver tumor promoting conditions. It is likely that an imbalance in nucleotide pools may have a significant role in the pathophysiology associated with these disorders.

Adenine Nucleotides↗
PubMed1994

Preliminary crystallographic analysis of a Fab specific for P-glycoprotein with and without bound peptide.

The antigen-binding fragment (Fab) of anti-peptide monoclonal antibody C219 raised against the multidrug resistance associated P-glycoprotein has been crystallized with and without bound peptide. The crystals of the Fab in the absence and presence of peptide belong to space groups P2(1) and P2(1)2(1)2(1), respectively. The volumes of both crystal forms are consistent with the presence of four Fab molecules per asymmetric unit. Diffraction data to 3.2 A resolution have been collected on a San Diego Multiwire Area Detector system from both crystal forms. Determination of the molecular replacement solutions is underway.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

An earlier proliferative response of hepatocytes in gamma-glutamyl transferase positive foci to partial hepatectomy.

One of the hallmarks of initiated hepatocytes is their resistance to several hepatotoxins. This property forms the basis for their selective growth under conditions which are inhibitory to the non-initiated hepatocytes. Selective growth of initiated hepatocytes also occurs, albeit at a low level, in initiated rat liver without exposure to any known promoting regimen and/or in the absence of any known selective pressure to which initiated hepatocytes can possibly be resistant. This latter phenotypic property of initiated hepatocytes was further characterized by comparing the kinetics of response of hepatocytes in gamma-glutamyl transferase positive foci and in the surrounding liver to 2/3 partial hepatectomy both in the presence and in the absence of a promoting regimen. Male Fischer 344 rats (130-150 g) were initiated with a single dose of diethylnitrosamine and 1 week later they were placed on either a semi-synthetic basal diet or a promoting diet containing 1% orotic acid. Partial hepatectomy was performed 15 weeks after initiation and animals from both groups were killed at 12, 16, 20, 24, 30, 36, 48, 72 or 96 h after operation. Each animal received a pulse of 3H-labelled thymidine 1 h prior to killing. Autoradiographic studies revealed that hepatocytes in gamma-glutamyl transferase positive foci in the livers of rats fed the basal diet were significantly labelled at 16 h post-partial hepatectomy while surrounding hepatocytes were still virtually quiescent (LI 12.7 +/- 4.7 versus 1.2 +/- 0.5%, respectively). Higher labelling index in foci compared to the surrounding liver was also seen at 20 h post-PH (36.9 +/- 2.6 versus 21.5 +/- 2.4). Similar earlier response of hepatocytes in gamma-glutamyl transferase positive foci was also seen in initiated rats exposed to dietary orotic acid. In addition, orotic acid treatment appears to have imposed a slight delay on the entry of hepatocytes in the surrounding liver into 'S' phase and thereby enhancing the differential of growth response between these two populations.

Animals↗
PubMed1994

Perturbations of endogenous levels of orotic acid and carcinogenesis: effect of an arginine-deficient diet and carbamyl aspartate on hepatocarcinogenesis in the rat and the mouse.

Feeding excess orotic acid (OA) in the diet promotes the carcinogenic process in different organs including the liver. A number of metabolic and genetic disorders are associated with increased synthesis of endogenous OA and some of these disorders appear to pose an increased risk of liver cancer development. This study therefore examines whether excess OA of endogenous origin also exerts a promoting effect on hepatocarcinogenesis in the mouse and the rat. Increased endogenous synthesis of OA was achieved by (i) feeding a diet deficient in arginine (AD) and (ii) feeding excess dietary carbamylaspartate (CA), a precursor for the synthesis of OA. A single dose of diethylnitrosamine (DENA) was given i.p. to male Fischer 344 rats (200 mg/kg) or to male DBA/2 mice (90 mg/kg). One week later they were placed on either AD diet or the same diet supplemented with 1.35% arginine (AS) for a total of 4 weeks. Two-thirds partial hepatectomy (PH) was performed at the end of the second week. All animals were then transferred to a control semisynthetic basal diet for a total of 20 weeks before they were killed. The results indicated that AD diet increased the incidence of hepatic nodules in both rats (percentage area occupied by nodules was 4.7 +/- 0.4 in the AD group compared to a control value of 0.7 +/- 0.5) and mice (4/10 mice had nodules > 5 mm diameter in the AD group while none in the AS group had such large nodules). In another experiment male Fischer 344 rats similarly initiated with DENA were exposed to either basal diet or basal diet containing 2% CA for 4 weeks coupled with PH performed at the end of the second week. This regimen was followed by 20 weeks of feeding basal diet to both groups. Rats given CA developed larger hepatic foci and nodules (0.84 +/- 0.56 mm3) compared to the control group, which was fed basal diet throughout the experiment (0.07 +/- 0.03 mm3). Further, both AD diet and dietary CA, like dietary OA, induced an increase in hepatic uridine nucleotides. Taken together, these results suggest that increased levels of endogenously synthesized OA, like exogenously supplied excess OA, can induce an imbalance in hepatic nucleotide pools and can exert a promoting effect on hepatocarcinogenesis.

Animals↗
PubMed1992

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.

Acetylcholine↗
PubMed1992

Abnormal hepatic nucleotide pools in sparse fur (spf) mutant mice deficient in ornithine transcarbamylase.

Sparse fur hemizygous male mice are over 90% deficient in ornithine transcarbamylase and exhibit increased synthesis of orotic acid. Because our earlier studies have demonstrated that orotic acid is a liver tumor promoter in the rat, it was of interest to determine whether this genetic disorder also increases the risk of tumor promotion. The results revealed that the livers of mutant mice showed a fourfold increase in uridine nucleotides and a 50% decrease in adenosine nucleotides compared to corresponding controls, a pattern of nucleotide pool imbalance similar to that seen in the livers of rats exposed to orotic acid under promoting conditions. Creation of such an imbalance appears to be important for orotic acid to exert its promotional effects. Sparse fur mutant mouse may, therefore, be an ideal animal model to study the tumor-promoting effects of orotate.

Animals↗
PubMed1992

[Mechanism of the effect of serine and threonine on ammoniagenesis and biosynthesis of orotate in the mouse].

We have evaluated the effects of serine and threonine on orotate metabolism in mice, by injecting i.p. 0.5 to 1.5 mmol/100 g of body weight, during 4 weeks of experimentation. The results indicate that serine as well as threonine cause a significant increase (p less than 0.01) of plasma ammonia and urinary orotate. We have also studied the effects of various inhibitors, adenine (0.3% diet), N-(phosphonoacetyl)-L-aspartate (PALA) (10 mg/100 g, i.p.), acivicin (1 mg/100 g, i.p.) and cycloheximide (1.5 mg/kg, i.p.) on orotate synthesis in mice injected with serine (2.5 mmol/100 g, i.p.) or threonine (1.25 mmol/100 g, i.p.). The results show an increase (p less than 0.05) of urinary orotate following an adenine-rich diet, and a reduction of orotate following PALA (p less than 0.01). The orotic aciduria remained insensitive to acivicin while it was inhibited by cycloheximide. These results suggest that adenine blocks the utilisation of orotate and consequently could affect the biosynthesis of pyrimidines; the induction of orotate by serine and threonine is controlled by the translation of a specific protein synthesis, and necessarily implicates the mitochondrial carbamyl phosphate synthetase-I (CPS-I). The regulating step in this synthesis of orotate could also be the transport of carbamyl-phosphate from the mitochondria to cytosol.

Adenine↗
PubMed1991

Expression and cell membrane localization of rat M3 muscarinic acetylcholine receptor produced in Sf9 insect cells using the baculovirus system.

A recombinant baculovirus bearing the cDNA coding for the rat muscarinic acetylcholine receptor subtype M3 placed under the control of the Autographa californica nuclear polyhedrosis virus polyhedrin gene promoter, was constructed. Polymerase chain reaction screening was used to identify the recombinant baculovirus. Northern blot analysis of total RNA from insect cells infected with the recombinant baculovirus indicated that the transcripts were abundant. Binding assays carried out with the muscarinic antagonist [3H]quinuclidinyl benzilate indicated that more than 1 x 10(6) receptors were produced per cell. Immunofluorescence microscopy showed that the receptor is located on the cell surface.

Acetylcholine↗
PubMed1991

Purification and functional characterization of the human beta 2-adrenergic receptor produced in baculovirus-infected insect cells.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

Adenylyl Cyclases↗
PubMed1990

Studies on the effect of dietary orotic acid on mouse liver carcinogenesis induced by diethylnitrosamine.

The present study was designed to determine whether orotic acid, a liver tumor promoter in the rat, also promotes liver carcinogenesis in the mouse. Eight-week-old male BALB/c mice were initiated with diethylnitrosamine (90 mg/kg i.p.). One week later they were divided into 2 groups and given either a basal diet or the basal diet containing 1% orotic acid (OA). They were killed at 6 or 10 months after the administration of the carcinogen. At 6 months, no nodular lesions were seen in mice whether or not they were exposed to OA. However by 10 months 100% of mice in both groups developed hepatic nodules. OA neither shortened the latent period for the appearance of the nodular lesions not did it increase the size of the nodules. Although BALB/c mice exhibited an increase in uridine nucleotides and a decrease in adenosine nucleotides in the liver upon exposure to OA, the magnitude of the change was less compared with that seen in the rat liver. The resistance of BALB/c mouse to the tumor-promoting effects of OA may reflect in part the resistance of the mouse to OA-induced nucleotide pool imbalance.

Animals↗
PubMed1990

Prevalence of placentally transmitted antibodies for measles in infants 3 to 11 months old in an urban slum community.

Upto 35% of infants aged between 6 and 11 months are infected with measles in India with its associated high morbidity and mortality. The objective of the study is to know the waning pattern of placentally transmitted antibodies (PTA) for measles so that the age at which children are likely to become susceptible to measles infection could be identified. A cross-sectional serological survey of children aged 3 to 11 months in one of the Integrated Child Development Service (ICDS) area in Madras city slums was done. Venous blood from 376 children was collected and was tested for Hemagglutination Inhibition (HI) antibodies by standard microtitration technique. Titre greater than or equal to 1:8 has been considered as protective. The proportion of children with immune level and the Geometric Mean Titre (GMT), declined to the least by 5 months which denotes that most of the infants become susceptible to measles infection from as early as 5 months of age. There is no significant difference in the waning pattern between different age groups, sex and nutritional status. A community study for effectiveness of measles vaccine at 6-8 months of age is needed to know the feasibility of immunization earlier than 9 months of age.

Age Factors↗
PubMed1988

Rapid and transient induction of c-fos, c-myc and c-Ha-ras in rat liver following glycine administration.

Administration of glycine (2.5 mmoles/100 g., i.p.) results in an increased expression of several cell cycle dependent genes such as c-fos, c-myc and c-Ha-ras in the rat liver. The increased expression could be noticed as early as 20-40 minutes and declined by 2 hours following glycine administration. The rapid rise and decline in the mRNA levels of c-fos, c-myc and c-Ha-ras in response to glycine is of significance because in response to a wide variety of growth stimuli, these proto-oncogenes exhibit a temporal sequence in their expression; for example, the expression of c-fos precedes that of c-myc, which in turn precedes the increased expression of c-Ha-ras. The experimental model using a simple amino acid such as glycine will be useful in exploring some of the mechanisms of regulation of expression of these proto-oncogenes.

Animals↗
PubMed1987

Dietary and metabolic manipulations of the carcinogenic process: role of nucleotide pool imbalances in carcinogenesis.

Perturbations in DNA and/or membranes are considered to be important for the carcinogenic process. A search for nutritional and metabolic means of disturbing the homeostasis of DNA and membranes revealed that nucleotide pools offer an exciting possibility. An imbalance in nucleotide pools can exert a two-pronged attack on both DNA and membranes. When given to rats, orotic acid, a precursor of pyrimidine nucleotides, results in an imbalance in nucleotide pools (an increase in uridine nucleotides and a decrease in inosine/adenine nucleotides), alterations in both DNA and membranes, and promotion of carcinogenesis in the liver initiated by chemical carcinogens. Agents such as adenine and allopurinol, which inhibit the metabolism of orotic acid and thereby decrease the formation of uridine nucleotides, and galactosamine, which traps uridine nucleotides, inhibited the promotional effects of orotic acid in the liver. These results suggested that orotic acid needs to be metabolized to uridine nucleotides and the creation of a subsequent imbalance in nucleotide pools is important for the promotional effects of orotic acid. To determine whether the creation of a nucleotide pool imbalance is a more general mechanism of tumor promotion, two lines of approach were investigated. One was to determine the effect of orotic acid on promotion of carcinogenesis in other organs, and the second approach was to determine how to induce nucleotide pool imbalances by means other than orotic acid administration. It is interesting to note that orotic acid promotes carcinogenesis in duodenum initiated by azoxymethane. Regarding the second approach, it became apparent that several metabolic disturbances result in increased orotic acid synthesis and alterations in nucleotide pools.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effect of glycine on the induction of orotic aciduria and urinary bladder tumorigenesis in the rat.

The mechanism by which amino acids increase the cellular levels of orotic acid (OA) was investigated. Administration of glycine (2.5 mmoles/100 g) to rats resulted in a 100-fold increase in urinary OA excretion, which was inhibited by pretreatment with cycloheximide or actinomycin D. The induction of OA synthesis from NH4Cl but not from carbamoylaspartate (CA) was inhibited by cycloheximide, indicating that the cycloheximide sensitive step was after the formation of ammonia and before the formation of CA. The glycine-stimulated OA synthesis was not inhibited by acivicin, a potent inhibitor of the cytosolic carbamoylphosphate (CP) synthetase, implicating the mitochondrial CP synthetase in supplying the CP for OA synthesis. Preliminary results indicated that cycloheximide did not inhibit glycine-induced urea synthesis to any significant extent. The results thus suggest that (i) the increased OA synthesis induced by glycine requires a transcription-translation dependent step and (ii) the regulatory step may be the transport of mitochondrial CP to cytosol and/or the synthesis of cytosolic CA. Attempts to determine whether increased exposure of urinary bladder to high concentrations of OA will influence bladder tumorigenesis revealed that chronic administration of glycine (2.5 mmoles/100 g, ip, daily, 5 days a week for 20 weeks) resulted in a 44% increased incidence of hyperplastic, preneoplastic, and neoplastic lesions. Some of these rats also exhibited stones in urinary bladders. The mechanism by which glycine induces tumorigenesis in the urinary bladder is currently being explored.

Ammonium Chloride↗
PubMed1987

Complementarity between two rat liver tumor promoters.

A delay in the exposure of initiated rats to orotic acid (OA) beyond a specific time frame results in a progressive loss of promotional effect in liver carcinogenesis. The current study was designed to ascertain whether the loss of promotional effect could be counteracted by pre-exposing the initiated animals to other rat liver promoting regimens such as a diet deficient in choline (CD). Male Fischer 344 rats (150 g) were initiated with diethylnitrosamine (200 mg/kg, ip); 1 week later they were given either a CD diet or a CD diet supplemented with choline for 5 weeks. Animals from these two groups were then fed either a 1% OA diet or the basal diet for another 20 weeks. The results indicated that the loss of OAs promotion efficacy from delaying the start of the promoting regimen can be counteracted by pre-exposing the initiated rats to a CD diet. Thus in rats exposed to OA from the first week of initiation, 7% of the liver developed as nodular areas, whereas only 0.8% of liver was nodular when OA feeding was delayed by 5 weeks. This loss was abolished when initiated rats were fed a CD diet for 5 weeks prior to feeding OA for 20 weeks. These results suggest that in a rat liver tumor promotion model, two tumor promoters, OA and CD, show some degree of complementarity when given sequentially.

Animals↗
PubMed1986

Direct communication of a pulmonary artery with left atrium--report of a case with balloon occlusion studies and successful correction: a case report.

A 10 year old cyanotic boy had direct communication between right pulmonary artery and left atrium. During cardiac catheterization, the catheter-tip balloon occlusion of this communication resulted in restoration of arterial oxygen saturation to normal levels, predicting that surgical interruption of the communication would be curative. Successful surgical correction was achieved by interruption of the communication.

Cardiac Catheterization↗