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Biomedical subjects

T K Ray

Publications and source records attributed to T K Ray.

At least 19 recordsLinked to original sources
PubMed2004

The annual risk of tuberculous infection in Orissa State, India.

SETTING: Rural and urban areas of eight selected districts of Orissa State, India. OBJECTIVES: To estimate the average annual risk of tuberculous infection (ARTI) in the state. DESIGN: A community-based, cross-sectional tuberculin survey was conducted among children aged 1-9 years residing in a sample of rural and urban areas of eight districts of Orissa State. A stratified sampling was adopted for selection of clusters. Highly trained designated tuberculin testers administered 0.1 ml (1 TU) of PPD RT 23 with Tween 80 by the Mantoux method to 10 626 children. Transverse tuberculin reaction sizes were measured about 72 h later by trained designated tuberculin readers. The number of children satisfactorily test-read was 10 191. RESULTS: Based on the frequency distribution of tuberculin reaction sizes, the average ARTI in the state was estimated at 1.7-1.8%. The children residing in urban areas were observed to be at a significantly higher risk of infection than those in rural areas. CONCLUSION: The study in Orissa State suggests high rates of transmission of tuberculous infection and calls to attention the need for intensification of concerted and sustained tuberculosis control efforts.

Age Distribution↗
PubMed1993

RNA from normal anterior endoderm/mesoderm-conditioned medium stimulates myofibrillogenesis in developing mutant axolotl hearts.

In the axolotl, Ambystoma mexicanum, a recessive cardiac lethal mutation causes an incomplete differentiation of the myocardium. Mutant hearts do not contain sarcomeric myofibrils nor do they beat. We have previously shown that normal anterior endoderm, medium conditioned by endoderm, or total RNA extracted from endoderm stimulates differentiation of mutant hearts in culture as indicated by the presence of organized myofibrils and rhythmic contractions of the "rescued" mutant heart tube. In this study, to get a more highly purified sample of the "active" molecule, RNA extracted from endoderm-conditioned medium and was assayed for its ability to promote myofibrillogenesis in mutant hearts. Mutant heart mesoderm responded to conditioned-medium RNA in a dose-dependent manner. Proteinase K treatment of the RNA did not affect inductive activity, while digestion with RNase A completely abolished the ability to rescue mutant hearts. Confocal laser scanning microscopy of immunostained, organ-cultured hearts revealed that mutant hearts contain reduced amounts of the sarcomeric protein tropomyosin in an amorphous distribution, whereas normal and corrected mutant hearts contain tropomyosin primarily in organized myofibrils.

Ambystoma↗
PubMed1988

Characteristics of the isolated apical plasmalemma and intracellular tubulovesicles of the gastric acid secreting cells: demonstration of secretagogue-induced membrane mobilization.

Separation of the gradient-purified gastric microsome into two membrane subfractions of distinct enzymatic and phospholipid composition has been achieved by mild SDS (0.033% w/v) treatment followed by sucrose gradient centrifugation of the pig and rabbit gastric microsomes. While the high-density membranes had all of the (H+,K+)-ATPase and K+-pNPPase activities and revealed a single major 100-kDa band on SDS-PAGE, the low-density membranes contained all of the 5'-nucleotidase and nearly all of the Mg2+-ATPase. In the present study, the low-density subfraction has been characterized to be derived from the apical membranes and the high-density one from the intracellular tubulovesicular membranes of the parietal cells. Such characterization was based primarily on sole dependency of the apical plasma membranes on the endogenous activator for (H+,K+)-ATPase activity, differential sensitivity of the activator (AF)-dependent and -independent (H+,K+)-ATPase on micromolar vanadate and Ca2+, specific vitamin B12 binding ability of the apical plasmalemma, phospholipid and protein profiles of the two membrane subfractions, and other parameters. The AF, mentioned previously, has recently been implicated as a cytosolic regulator of the gastric (H+,K+)-ATPase [Bandopadhyay et al. (1987) J. Biol. Chem. 262, 5664-5670]. Two different forms (i.e., AF-dependent and -independent forms) of the (H+,K+)-ATPase are suggested to be present in the tubulovesicles on the basis of differential vanadate sensitivity while the AF-dependent form alone is present in the apical membranes. The data have been discussed in terms of stimulation-induced membrane transformation characteristic of the H+-secreting epithelia including the acid-secreting cells of the stomach.

Adenosine Triphosphatases↗
PubMed1988

Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior.

Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions.

4-Nitrophenylphosphatase↗
PubMed1988

A direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase.

1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of 32P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, 32P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of 32Pi to 32P-labeled 1-acyl-sn-glycerol-3-phosphate.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1987

Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa.

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed1987

Characteristics of a pure endogenous activator of the gastric H+,K+-ATPase system. Evaluation of the role as a possible intracellular regulator.

An endogenous activator capable of stimulating the gastric H+,K+-ATPase activity has been purified to homogeneity from dog and pig gastric cells and found to be a dimer of two identical 40-kDa subunits in the active state. Identical nature of the activator monomers was revealed by the detection of lysine as the sole N-terminal amino acid. The activator from one species can stimulate the H+,K+-ATPase from another species and vice versa. Such cross-activation is consistent with the striking similarities in the amino acid composition between the two species, suggesting considerable homology in the activator molecules from different species. The activator exhibited several unique features during modulation of the H+,K+-ATPase reaction. It appreciably enhances affinity of the H+,K+-ATPase for K+, known to increase turnover of the enzyme. To complement this K+ affinity, the activator also enhances ability of the H+,K+-ATPase to generate more transition state (E*.ATP) complex by increasing the entropy of activation (delta S++) of the system as revealed from an Arrhenius plot of the data on temperature activation. In addition, the activator shows both positive cooperativity and strong inhibition, depending on its concentration. Thus, up to the ratio of the H+,K+-ATPase and activator of about 1:2 (on the protein basis), the activator shows sigmoidal activation (Hill coefficient = 4.5), but beyond such concentration a strong inhibition was observed. Finally, Ca2+ at low (2-4 microM) concentration strongly inhibits the activator-stimulated H+,K+-ATPase. It is proposed that the activator may be acting as a link in the signal transducing cascade system between the intracellular second messenger (Ca2+) and the physiological response (gastric H+ transport).

Adenosine Triphosphatases↗
PubMed1987

Reaction mechanism of the gastric H+ +K+-dependent ATPase. Effects of inhibitor and pH.

The effect of nolinium bromide [2-(3,4-dichlorophenylamino)quinolizium bromide], which acts as a K+ antagonist in the gastric H+ +K+-dependent ATPase reaction, was investigated at the level of 32P-labelled intermediates of the gastric ATPase reaction. A concentration-dependent effect of nolinium bromide was observed on the concentrations of phosphorylated intermediates. At low (up to 50 microM) concentrations the drug did not interfere with the concentrations of intermediates but exhibited a competition with K+ at the level of both 32P-labelled intermediates and hydrolysis of ATP at pH 7.0. Similar competition was noted in the H+ +K+-dependent ATPase reaction. Low nolinium bromide concentrations also drastically slowed the enzyme turnover. The concentrations of the intermediates were lowered appreciably between 50 microM- and 100 microM-nolinium bromide without affecting the ATP hydrolysis, and the effects were independent of pH. Similar to the effects at pH 7.0, the drug also exhibited competition with K+ in lowering the E approximately P concentration at pH 5.0. A dramatic effect of pH on the K+-sensitivity as well as on turnover of the 32P-labelled intermediates was observed. Although the concentrations of intermediates remained nearly unaltered at various pH values, the K+-stimulated hydrolysis of ATP showed an optimum at pH 7.0 with sharp declines at pH 5 and 8. The data suggest a critical involvement of H+ in the conversion of the K+-insensitive E1 approximately P into the K+-sensitive E2 approximately P form of the enzyme. Nolinium bromide appears to function as a K+ analogue and seems to block the entry of K+ at the K X E2 step, thereby interfering with the enzyme turnover.

Adenosine Triphosphatases↗
PubMed1987

Acylation of glycerol 3-phosphate is the sole pathway of de novo phospholipid synthesis in Escherichia coli.

The inhibition of phospholipid synthesis engendered by starving glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli (plsB or gpsA) for G3P is incomplete; 5 to 10% of the normal rate of phospholipid synthesis remains, even after prolonged starvation. We report that G3P starvation of a strain having lesions in both the gpsA and plsB genes resulted in essentially complete (greater than 98.5%) inhibition of phospholipid synthesis, indicating that all de novo glycerolipid synthesis in E. coli proceeds by acylation of G3P.

Acetates↗
PubMed1986

NMR studies of pig gastric microsomal H+,K+-ATPase and phospholipid dynamics. Effects of ethanol perturbation.

The effects of ethanol on the gastric H+,K+-ATPase activity and the degree of mobility of various microsomal phospholipids were assessed using 31P and 1H NMR. This illuminated the role of lipid-protein association in the function of pig gastric microsomes. Treatment of gastric microsomes with 15% ethanol for 1 min at 37 degrees C inactivated the H+,K+-ATPase activity, which could largely be reconstituted by supplementation with phosphatidylcholine isolated from the gastric microsomes. Under similar conditions, the 1H NMR profile of the microsomal +N(CH3)3 choline moiety showed dramatic enhancement of peak intensity as well as a break point at 25 degrees C which was restored to the untreated control value after reconstitution. This break, together with the dramatic enhancement in the overall lipid profile, compared to the control and reconstituted microsomes, suggested a greater degree of freedom of movement of the microsomal lipids following ethanol perturbation. The data demonstrate the unique ability that a combined approach using 31P and 1H NMR holds as a noninvasive probe to study the structure-function relationship of biomembranes.

Adenosine Triphosphatases↗
PubMed1986

Regulation of insulin receptor activity of human erythrocyte membrane by prostaglandin E1.

Incubation of human erythrocyte membrane with low concentration of prostaglandin E1 or prostacyclin increased the binding of 125I-labeled insulin to the membrane. The binding of the radioiodinated hormone was maximally stimulated at 3 nM prostaglandin E1 and the use of higher concentrations (above 8 nM) of the autacoid tended to reverse its own effect at lower concentrations. While prostaglandins A1, A2, B1, B2, D2, F1 alpha, F2 alpha or 6-keto-prostaglandin F1 alpha had no effect on the binding of insulin to the erythrocyte membrane, prostaglandin E2 at similar concentrations decreased the binding of the hormone. The effect of prostaglandin E1 on the increased binding of the insulin was found to be reversible and depended on the occupancy of the autacoid molecules on the membrane and showed positive cooperativity. Scatchard analysis of the binding of 125I-labeled insulin to the erythrocyte ghosts indicated that in the presence of the autacoid, the binding capacity of the insulin receptor increased 2-fold (from 207 to 424 fmol/mg protein) without any change in the ghosts affinity for the ligand (Kd 2.4 X 10(-9) versus 2.49 X 10(-9) M). As a consequence of increased binding of insulin to the erythrocyte membrane in the presence of prostaglandin E1 (3.0 nM), the optimal concentration of the peptide hormone for the maximal reduction of the membrane microviscosity decreased from approx. 1.6 to approx. 0.4 nM. Addition of prostaglandin E1 alone at the above concentration to the assay mixture had no effect on the membrane microviscosity.

Alprostadil↗
PubMed1986

Mechanism of gastric antisecretory effect of thiocyanate: further evidence for the thiocyanate-induced impediment in gastric H+,K+-ATPase function.

Two hypotheses have recently been proposed for the thiocyanate inhibition of gastric acid secretion--a protonophore mechanism and an uncoupling model. The mechanistic aspects for the latter scheme have been examined on the following basis: capability of generating verifiable predictions, supporting evidence that is unambiguous, and compatibility with experimental realities. Gastric microsomes bind 5 nmol of SCN-/mg, and a "pure" and highly active fraction of H+,K+-ATPase prepared from gastric microsomes binds about 15 nmol of SCN-/mg. The affinity of SCN- binding to gastric microsomes changes from 10 to 25 mM in the presence of 20 mM K+ suggesting competition between K+ and SCN-. Potassium also displaces the bound SCN- from "pure" H+,K+-ATPase with a Ki of about 25 mM. Of the cations tested--Tl+, K+, Rb+, Cs+, NH4+, Na+, and Li+--Tl+ was the most effective in displacing bound SCN- while Na+ and Li+ were without effect. The effects of anions such as Cl-, NO3-, and gluconate were found to be nonspecific and absolutely dependent on K+ as cocation. Sulfate and OCN-, on the other hand, showed an ability to displace SCN- as both K+ and Na+ salts. For SO4(-2) the K+ form was much more effective than the Na+ salt. Besides these antagonistic effects of K+ and congeners with the H+,K+-ATPase-bound SCN-, a competition between K+ and SCN- was also observed at the level of gastric K+-stimulated pNPPase reaction. The effects of SCN- and two other unrelated anions, F- and NO2-, on artificial delta pH across the microsomal vesicles exhibited a lack of appreciable change up to 5 mM and a small (about 13%) reduction between 10 and 20 mM. However, a combination of CCCP and nigericin or valinomycin completely abolished the delta pH under identical conditions. The present data in conjunction with other reports suggest that the proton impediment model best explains the gastric antisecretory effects of SCN-.

4-Nitrophenylphosphatase↗
PubMed1986

Prostacyclin stimulation of the activation of blood coagulation factor X by platelets.

When platelets were incubated with prostacyclin, prostaglandin E1, or prostaglandin D2 at concentrations insufficient to increase the level of adenosine 3',5'-monophosphate (cyclic AMP), coagulation factor X was activated by a platelet cysteine protease. Prostacyclin or prostaglandin E1 at higher concentrations increased the cyclic AMP level and inhibited the activation of factor X by platelets. Inhibition of platelet adenylate cyclase by 2',5'-dideoxyadenosine allowed the activation of the protease at higher concentrations of the autocoids. Prostaglandins A1, A2, B1, B2, E2, F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, which do not affect platelet cyclic AMP level, did not stimulate the protease.

Adenylyl Cyclases↗
PubMed1986

K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H+ + K+)-transporting ATPase. Evidence supporting a new model for the univalent-cation-transporting ATPase systems.

Studies with intact and lysed gastric microsomal vesicles demonstrate that there are two pNPP (p-nitrophenyl phosphate)-and one ATP-hydrolytic sites within the gastric H+, K+-ATPase [(H+ + K+)-transporting ATPase] complex. Whereas the ATPase site is located exclusively on the vesicle exterior, the pNPPase sites are distributed equally on both sides of the bilayer. Competition by ATP for the pNPPase reaction on the vesicle exterior suggests that both ATP and pNPP are hydrolysed at the same catalytic site present at the outside surface of the intact vesicles. However, a biphasic inhibition of the K+-pNPPase (K+-stimulated pNPPase) by ATP in the lysed vesicles suggest the pNPPase site of the vesicle interior to have very low affinity (Ki approximately equal to 1.2 mM) for ATP compared with the vesicle exterior (Ki approximately equal to 0.2 mM). Studies with spermine, which competes with K+ for the K+-pNPPase reaction without inhibiting the H+, K+-ATPase, suggest there are two separate K+ sites for the pNPPase reaction and another distinct K+ site for the ATPase reaction. In contrast with the K+ site for the ATPase, which is located opposite to the catalytic site across the bilayer, both the K+ and the catalytic site for the pNPPase are located on the same side. The data clearly demonstrate that the pNPPase is not a manifestation of the phosphatase step of the total H+, K+-ATPase reaction. The K+-pNPPase associated with the Na+, K+-ATPase also has properties strikingly similar to the gastric K+-pNPPase system, suggesting a resemblance in the basic operating principle of the two ion-transporting enzymes. A unified model has been proposed to explain the present data and many other observations reported in the literature for the ATPase-mediated transport of univalent cations.

4-Nitrophenylphosphatase↗
PubMed1986

Purification and characterization of a cytosolic activator protein for the gastric H+,K+-ATPase system from dog fundic mucosa.

An endogenous protein activator (AF) responsible for the activation of the gastric H+,K+-ATPase system, identified recently as the biochemical mechanism for the transport of H+, has been purified to homogeneity and partially characterized. The purification procedure (at 0-4 degrees C) involves simultaneous concentration and dialysis of the cytosolic fraction from dog fundic cells under negative pressure, pH 4.8 precipitation and two consecutive gel filtration steps on sephacryl S-200 columns. The highly purified and active AF is a protein of 80 Kd consisting of two identical subunits of 40 Kd each. The AF not only stimulates the gastric H+,K+-ATPase activity but also greatly enhances the rate of ATPase dependent proton pumping inside gastric microsomal vesicles. The data clearly suggest an important regulatory role of the cytosolic AF in the gastric HCl secretory process.

Adenosine Triphosphatases↗
PubMed1985

Control of erythrocyte membrane microviscosity by insulin.

The human erythrocyte membrane binds insulin through high-affinity, low-capacity binding sites (dissociation constant Kd1 2.45 X 10(-9)M; capacity n1 207 fmol/mg protein) and low-affinity, high-capacity binding sites (Kd2 0.63 X 10(-6) M; n2 37 pmol/mg protein). Treatment of the erythrocyte membrane or the intact cells with the physiological concentration of insulin, which is within the range of Kd value of the high-affinity sites, results in a significant reduction of the membrane microviscosity and the filtration time of the intact cells. Use of supraphysiological concentrations of the hormone reverses the effect of the lower concentration of insulin on the membrane microviscosity and the filtration time.

Erythrocyte Membrane↗