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Biomedical subjects

V Sitaramam

Publications and source records attributed to V Sitaramam.

At least 19 recordsLinked to original sources
PubMed1997

Volume regulation of spermatozoa by quinine-sensitive channels.

Bovine spermatozoa were shown to exhibit rapid regulatory volume decrease (RVD) when exposed to hypotonic saline media. This quinine- and quinidine-sensitive regulatory volume decrease was coincident with K+ release due to stretch-activation of inhibitor-specific presumptive K+ channels. The regulatory volume decrease response was much faster than a similar phenomenon observed in human peripheral blood lymphocytes. Studies on volume changes in different electrolyte and nonelectrolyte media suggested that: (1) this inhibitor-specific channel could also be a nonspecific pore in the spermatozoal membrane for nonelectrolytes below 150 daltons; (2) subpopulations (of nearly equal size) of the spermatozoa differ in the expression of the pore; (3) capacitation abolishes this distinction between subpopulations of spermatozoa; and (4) the general case of RVD for other mammalian spermatozoa was also established.

Animals↗
PubMed1996

Assessment of molecular sieving across bacterial outer membrane of Pseudomonas.

The role of the permeability barrier of the outer membrane of Pseudomonas was re-evaluated based on the physical theory of molecular sieving in view of its intrinsic antibiotic resistance. We developed a set of analytical procedures based on parametric and non-parametric statistical tests to evaluate, validate and adopt the better among a set of competing non-linear models of diffusion. The molecular mass dependence of uptake of non-electrolytes in bacteria yielded a quantitative measure to distinguish between sieving mechanisms and specific uptake/efflux mechanisms. The experimental data, supported by the physical model of DEAE-Sephadex and various analytical models and extensive simulation of the errors, both in measurement and models, yielded evidence consistent with the relaxation of the outer membrane matrix barrier in Pseudomonas.

Carbohydrate Metabolism↗
PubMed1995

Influence of osmolality of the medium on photosynthetic electron transport, proton fluxes and photophosphorylation in isolated thylakoids.

The high osmotic potential inhibition of photosynthetic electron transport was determined to be related to membrane compaction rather than to an effect of primary thylakoid volume changes. Osmotic inhibition of proton fluxes and phosphorylation were entirely due to osmotic inhibition of electron transport. The ATPase activity, the nature of coupling and the rate constant of proton efflux were not influenced by osmotic pressure, while the rate constant and the extent of proton influx were inhibited by osmotic pressure.

Culture Media↗
PubMed1994

Stretch sensitivity of transmembrane mobility of hydrogen peroxide through voids in the bilayer. Role of cardiolipin.

Availability of voids for diffusion of quinone in the membrane was shown to be the rate-limiting step in electron transport in mitochondria and chloroplasts (Mathai, J. C., Sauna, Z. E., John, O., and Sitaramam, V (1993) J. Biol. Chem. 268, 15442-15454). The primary role of voids in these diffusion-controlled reactions required a more rigorous documentation of the role of diffusion in membranes by independent measurements. The transbilayer diffusion of hydrogen peroxide as monitored by occluded catalase activity was developed as a kinetically valid probe to specifically address this question. This in turn led to unique results on the mechanistic basis of stretch (= hypo-osmotic) activation of hydrogen peroxide permeation via such voids. The rate of peroxide permeation is shown to be markedly stretch sensitive in some cells/organelles (e.g. peroxisomes) and insensitive in others (e.g. erythrocytes); this was equally true of liposomes prepared from lipids extracted from the corresponding cells/organelles. The molecular basis of stretch sensitivity was uncovered using specific binary mixtures of lipids: while pure phosphatidyl choline liposomes were stretch insensitive, these became sensitive when doped only with specific lipids, viz. cardiolipin and cerebrosides. Cholesterol abolished this stretch sensitivity in ternary mixtures. Induction of stretch sensitivity by cardiolipin was marked by lowering of activation energy for peroxide diffusion, a negative temperature coefficient for glucose permeation while further addition of cholesterol reversed these phenomena. The steady state fluorescence polarization studies revealed intimate correlations between anisotropy, hydrogen peroxide diffusion, and stretch sensitivity consistent with presence of voids in these binary mixtures.

Animals↗
PubMed1993

Rate-limiting step in electron transport. Osmotically sensitive diffusion of quinones through voids in the bilayer.

Respiration in mitochondria and photosynthesis in chloroplasts varied with the osmotic stretch of the membrane such that these processes were uniformly inhibited at higher osmolalities. A systematic evaluation of segmental electron transport in these intact particles showed that no individual complex exhibited osmotic sensitivity, whereas osmotic sensitivity appeared wherever the assay involved crossing over the corresponding quinone in the electron transport chain. The evidence was consistent with the rate-limiting step in electron transport being the availability of voids for quinone migration rather than any of the components of electron transport chain per se. Evidence based on quinone reconstitution in mitochondria depleted of quinone by acetone treatment clearly distinguished the kinetic control in the hypotonic domain and diffusive control via availability of voids in the hypertonic domain. Influence as well as the presence of voids was further confirmed in quinone-depleted mitochondria reconstituted with quinone as well as cholesterol. Decrease in lateral diffusion of the fluorescent probe, 12-(9-anthroyl)stearic acid, on osmotic compression of the bilayer is consistent with a change in void size distribution on osmotic compression of the bilayer. A direct correlation between succinate cytochrome c oxidoreductase activity and diffusivity of fluorescent probe 12-(9-anthroyl)stearic acid confirmed the availability of voids as the rate-limiting step in electron transport.

Acetone↗
PubMed1992

Charge anisotropy across biological membranes: evidence and implications.

Membrane proteins exhibit charge anisotropy across the bilayer with the vector positive inwards. The proton pumps, primary or secondary, which have been examined as a subset of these membrane proteins, also reveal charge anisotropy based on their sequence data. The direction of the anisotropy appears to satisfy the observed directional gradient of protons mediated by these proteins. A correct description of transport requires attention to local as well as field effects of the charge anisotropy of membrane proteins.

Anisotropy↗
PubMed1992

Statistical testing of equality of two break-points in experimental data.

Two examples in quantitative biology are examined to emphasize the need for two-phase regression models: the osmotic behaviour of cells and the non-linear temperature kinetics of membrane-bound enzyme systems. Existing statistical techniques are inadequate to test the equality of break-points of two data sets for specific reasons. We suggest here a pragmatic solution by way of a computer programme useful in applying two-phase regression models to such data sets wherein a decision needs to be made whether the critical transition differs or not.

Algorithms↗
PubMed1992

Membrane instability in respiring mitochondria: role of phosphate.

Metabolically-induced (spontaneous) high amplitude swelling of mitochondria has been shown to be due to a serial disruption of the mitochondrial membranes [D. Sambasivarao & V. Sitaramam (1985), Biochim Biophys Acta, 806, 195-209]. Phosphate- and arsenate-induced swelling was investigated in mitochondria to evaluate the role of phosphate transport in the instability created in the mitochondrial membranes. Phosphate-induced swelling in respiring mitochondria was similar to spontaneous swelling. Both represent essentially colloidal swelling due to the variable porosity induced in the inner membrane to polyols by respiration. Swelling of non-respiring mitochondria at high ammonium phosphate concentrations was, on the other hand, primarily due to high permeability to phosphate. This membrane instability created by phosphate transport in the surrounding lipid involves neither the endogenous nor the exogenous Ca2+.

Animals↗
PubMed1991

Characterization of mitochondrial membrane fragments resulting from spontaneous swelling: novel stimulation of NADH-dependent respiration by carboxylic acids.

Metabolically induced high amplitude swelling of rat liver mitochondria has been found to result in the formation of a heterogeneous population of mitochondrial membranes consisting of right side-out particles with occluded fumarase activity and inside-out particles/fragments capable of NADH-dependent respiration. This rotenone-sensitive, uncoupler-insensitive, NADH-dependent respiration was specifically and instantaneously stimulated by several ligands such as glutamate and malate (which can be metabolized) and, interestingly, even lactate (which could not be metabolized by the swollen mitochondria). These observations suggest that high amplitude swelling results in a novel type of control of respiration in these fragments.

Animals↗
PubMed1991

NADH-dependent respiration in osmotically inactive swollen mitochondria: does transport replace phosphorylation in mediating respiratory control in swollen mitochondria?

Rotenone-sensitive, uncoupler-insensitive, NADH-dependent respiration was demonstrated in osmotically inactive fragments of the mitochondrial inner-membrane obtained following high amplitude (spontaneous) swelling. This NADH-dependent respiration as well as mitochondrial ATPase activity was stimulated by ligands which are known to be transported by specific transporters/mechanisms. The ligands capable of this anomalous respiratory control included several intermediates of the citric acid cycle, besides non-metabolizable ligands including lactate, cations such as K+ and Ca2+. The interaction between NADH-dependent respiration and these ligands, as manifested by stimulation of respiration, was strongly ionic strength-dependent. The thermodynamic relationship between respiratory control and stimulation of transport ATPase by the relevant transportable ligands could also be demonstrated in the conventional (rat liver) microsomes. These experimental results offer a novel experimental base for search into an intra-membranous mechanism of energy transduction.

Animals↗
PubMed1989

Variable porosity of the mitochondrial inner membrane induced by energization.

The empirically observed relationship between the activity of membrane-bound enzyme systems and transporters and the external osmotic pressure offered a direct method to assess the reflection coefficients to polyols in respiring mitochondria. These osmotically modulated reaction rates varied with the molecular mass of the external polyol similar to volume and solute fluxes across dialysis membranes. The equivalent pore radii of mitochondria were shown to increase with respiration (and temperature) and decrease on addition of the uncoupler, 2,4-dinitrophenol. The magnitude of the induced porosity in the inner membrane was large enough to render the chemiosmotic mechanism inoperable in well-coupled rat liver mitochondria.

2,4-Dinitrophenol↗
PubMed1989

Differential effects of osmotic pressure on mitochondrial respiratory chain and indices of oxidative phosphorylation.

Oxidative phosphorylation was critically evaluated in terms of activities which are sensitive and insensitive to variations in external osmotic pressure in mitochondria. Integrity of mitochondria was determined in terms of a variety of parameters, including the latency of the occluded enzymes, by careful titrations as a function of external osmotic pressure as well as detergent concentrations. The evidence indicated that the rate-limiting step in respiratory states 2 and 4 would be osmotically insensitive, as opposed to the osmotically sensitive respiration of states 1 and 3 and uncoupler-stimulated respiration with glutamate + malate and succinate. Cytochrome oxidase activity in mitochondria as well as in purified reconstituted systems exhibited osmotic insensitivity but marked sensitivity to ionic strength, offering an interesting model to study the osmotically insensitive respiration. Cytochrome oxidase activity led to permeation of mannitol across the mitochondrial inner membrane. Stimulation of cytochrome oxidase activity by uncouplers did not require an intact membrane.

2,4-Dinitrophenol↗
PubMed1989

Genetic code preferentially conserves long-range interactions among the amino acids.

The physical properties of amino acids were investigated in order to evaluate their possible relationship to the assignment of codons for amino acids in the genetic code. A comparison of the interconversion probability between amino acids and the distances between the amino acids for individual physical properties revealed a striking hierarchy among the physical properties. Surprisingly, it is the long-range/solvent interactions and not the short-range/stereochemical properties which are preferentially conserved in the genetic code.

Amino Acids↗
PubMed1988

ATP hydrolysis induces variable porosity to mannitol in the mitochondrial inner membrane.

Osmotic titration of ATPase activity in rat liver mitochondria was consistent with enhanced porosity of the mitochondrial inner membrane to mannitol due to ATP hydrolysis even when endogenous respiration was inhibited by rotenone. The occluded ATPase activity, which exhibits osmotic activation with an optimum near isotonicity, depends both on the ATPase activity per se and on the activity of the ADP/ATP carrier. Purified ADP/ATP carrier incorporated into small, unilamellar liposomes was critically shown to exhibit dependence of its activity on the osmotic pressure differences across the membrane, with maximal activity corresponding to isotonicity, regardless of the actual internal tonicity.

Adenosine Triphosphate↗
PubMed1987

Diagnostic and prognostic value of RNA-proteolipid in sera of patients with malignant disorders following therapy: first clinical evaluation of a novel tumor marker.

The circulating level of a novel RNA-proteolipid complex associated with malignant diseases was critically evaluated as a tumor marker in clinical oncology. The complex, isolated from the sera of cancer patients, exhibited unvarying chemical composition regardless of the cell type and clinical staging. Clearance from blood was rapid with a half-life of approximately 2 days. Tumor mass could be correlated with the circulating level. After effective treatment the level fell and rose again 10 months prior to the conventional clinical diagnosis of relapse.

Biomarkers, Tumor↗
PubMed1986

Anomalous permeability and stability characteristics of erythrocytes in non-electrolyte media.

The permeability characteristics of the erythrocyte membrane were critically evaluated in electrolyte and non-electrolyte (sucrose) media by ion-selective electrodes and radioactive polyol fluxes as well as by the novel technique of osmometry. K+ efflux demonstrated a linear osmotic susceptibility distinct from Na+ influx upon incubation in NaCl media of various tonicities. In non-electrolyte media, acidification of the medium, large fluxes of K+, sucrose and even haemoglobin (as manifest by hypertonic disruption) were consistent with enhanced porosity of the bilayer due to the field created by surface charge density leading to density fluctuations in the bilayer.

Animals↗
PubMed1986

Enzyme-linked immunolysis assay for tumour-specific surface antigens.

Expression of the activity of the cytosolic enzyme lactate dehydrogenase released on cell lysis by tumour specific antibodies in the presence of complement was investigated by direct assay of the expression of the occluded enzyme in tumour cell suspensions. This method, called Enzyme Linked Immunolysis Assay (ELILA) was shown to be more sensitive than conventional cytotoxic assays. The data fit well in the linear regression model, so that the technique can be used for quantitation of the antibody titres.

Animals↗