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Biomedical subjects

V V Kumar

Publications and source records attributed to V V Kumar.

16 recordsLinked to original sources

Prevalence and distribution of the hmw and hia genes and the HMW and Hia adhesins among genetically diverse strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. In previous work we identified high-molecular-weight adhesins referred to as HMW1 and HMW2, expressed by nontypeable strain 12, and determined that most strains of nontypeable H. influenzae express one or two antigenically related proteins. More recently, we determined that some strains lack HMW1- and HMW2-like proteins and instead express an adhesin called Hia. In the present study, we determined the prevalence and distribution of the hmw and hia genes in a collection of 59 nontypeable strains previously characterized in terms of genetic relatedness. Based on Southern analysis, 47 strains contained sequences homologous to the hmw1 and hmw2 genes and nine strains contained homologs to hia. No strain harbored both hmw and hia, and three strains harbored neither. Although the hmw and hia genes failed to define distinct genetic divisions, the hmw-deficient strains formed small clusters or lineages within the larger population structure. Additional analysis established that the IS1016 insertion element was uniformly absent from strains containing hmw sequences but was present in two-thirds of the hmw-deficient strains. As IS1016 is associated with the capsule locus (cap) in most encapsulated strains of H. influenzae, we speculate that hmw-deficient nontypeable strains evolved more recently from an encapsulated ancestor.

Adhesins, Bacterial

Lipid molecular shapes and membrane architecture.

A simple biomembrane like erythrocyte contains well over hundred lipid species with diverse molecular shapes differing in the number of acyl chains, chain length, unsaturation and head group composition. A delicate balance between these molecular shapes is necessary in order to have a functional membrane. It is well established that the activities of a number of membrane-bound enzymes and other properties such as aggregation, spontaneous vesiculation, pathophysiological properties and lipid-protein interactions of lipids depend on the acyl chain length, unsaturation and head group composition. In fact, the molecular shape of a phospholipid molecule, as modulated by changes in chain length, unsaturation and head group composition, is probably what is affecting the above mentioned properties. The molecular shape of a lipid depends on a dimensionless packing parameter, S, the value of which influences the size and shape of aggregate formed upon hydration. In fact, the additivity of S values of lipid mixtures explains a number of experimental observations. The molecular shape concept, although very simple, explains many membrane phenomena like complementarity of molecular shapes of non-bilayer lipids to form stable bilayers. Membrane permeability is controlled to a large extent by lipid packing which depends upon molecular shapes. In fact, membranes maintain their lamellar structure by delicately balancing the composition of bilayer-forming and non-bilayer-forming lipids indicating that complementarity of molecular shapes is essential to maintain the permeability barrier. Based on this, the complementary molecular shape model of cell membrane is proposed.

Animals

Phosphatidyl-Tris rather than N-acylphosphatidylserine is synthesized by Rhodopseudomonas sphaeroides grown in Tris-containing media.

We have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, 13C NMR, and 1H NMR spectra. Comparison of these data with those reported for a phospholipid isolated from Rhodopseudomonas sphaeroides grown in Tris-supplemented media [Donohue et al. (1982) Biochemistry 21, 2765-2773] indicates that R. sphaeroides synthesizes phosphatidyl-Tris rather than N-acyl-PS.

Acylation

Complementary molecular shapes and additivity of the packing parameter of lipids.

Physical dimensions of a membrane component influence its phase preference upon hydration. A dimensionless packing parameter, S, given by S = V/al, where V is the hydrocarbon volume, a is the area of the head group, and l is the critical length of the hydrocarbon chain, is useful in determining the phase preference of a lipid, and the value of S usually lies between 0.5 and 1 for bilayers. Here, the value of S is calculated for phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) as a function of chain length, and it is shown that diacylPC having an S value of less than 0.74 does not form bilayers. For example, diacylPC, up to a chain length of eight carbon atoms, forms only micelles, whereas higher homologs with S greater than 0.74 form bilayers. It is also shown that when lipid molecules having complementary shapes associate, the value of S becomes additive. Using the additivity of S, a number of experimental results for lipid mixtures can be explained. For example, lysoPC and cholesterol form lamellar structures between 45 and approximately 80 mol% cholesterol, and the additive value of S for this region is between 0.74 and 1. Similarly, the additivity of S shows that the maximum amount of cholesterol that can be incorporated into PC bilayers is 50 mol%, in agreement with experimental studies.

Cholesterol

Lanthanide-induced phosphorus-31 NMR downfield chemical shifts of lysophosphatidylcholines are sensitive to lysophospholipid critical micelle concentration.

Lysophosphatidylcholine (lysoPC) monomers or micelles in water give rise to a narrow, isotropic phosphorus-31 NMR signal (40.6 ppm; v1/2 1.7 Hz; 32.2 MHz). Upon addition of praseodymium ions, the phosphorus signals are shifted downfield. However, the downfield shifts for the longer-chain lysophosphatidylcholines, which exist in the aggregated state, are far greater than those for the shorter-chain homologues, which exist as monomers. At a Pr3+/lysoPC molar ratio of 0.5, the signals of C12lysoPC through C18lysoPC were shifted by 12.1 ppm, whereas the signals of C6lysoPC and C8lysoPC were shifted by only 2.26 ppm. This very pronounced difference in lanthanide-induced downfield shifts between micelles and monomers can be utilized to determine with accuracy lysoPC critical micelle concentrations (CMC) from downfield shift-vs.-concentration plots. The CMC values we determined were 57 mM for C8lysoPC, 5.7 mM for C10lysoPC, and 0.6 mM for C12lysoPC. The shift reagent phosphorus-31 nuclear magnetic resonance technique particularly lends itself to the measurement of CMC values in the millimolar and high micromolar range. The method can equally be used for measuring critical micelle concentrations of short-chain phosphatidylcholines.

Kinetics

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy.

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First, rac-1(3)-O-alkylglycerol is prepared by alkylation of rac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine gives rac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A2 (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The "unnatural" 3-O-alkyl-2-O-acyl-sn-glycero-1-phosphocholine enantiomer, which is not susceptible to phospholipase A2 cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.

Acylation

Lipids in gap junction assembly and function.

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions. The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

Animals

Lysophosphatidylcholine stabilizes small unilamellar phosphatidylcholine vesicles. Phosphorus-31 NMR evidence for the "wedge" effect.

Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.

Lipid Bilayers

Asymmetry of lysophosphatidylcholine/cholesterol vesicles is sensitive to cholesterol modulation.

Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol % chol, freeze-fracture EM showed homogeneous populations of small unilamellar vesicles averaging 260-310 A in diameter. Phosphorus-31 nuclear magnetic resonance was used to characterize the stacklike lamellae and to measure the distribution of the lysophospholipid between the outer and inner leaflet of the vesicles as a function of sterol concentration. We found that in lysoPC/chol dispersions containing less than equimolar amounts of cholesterol (25-47 mol %), the entire phosphorus signal (40.5 ppm) was shifted downfield by 10.5 ppm upon addition of Pr3+ (2.4 mM), consistent with the stacklike lamellar structures in which all lysoPC head groups are accessible to the ions. By contrast, addition of Pr3+ to lysoPC/chol vesicles containing equimolar or higher amounts of cholesterol (up to 60 mol %) gave rise to two phosphorus peaks. The more intense downfield signal (51.0 ppm) responsive to paramagnetic ions was assigned to lysoPC located in the outer vesicle leaflet. The upfield signal (40.5 ppm), which was not affected by the ions, was assigned to inside lysoPC. For lysoPC/chol (1:1) vesicles, an outside to inside lysophospholipid ratio (Ro/i) of 6.5 was determined. Essentially the same Ro/i value (6.7) was obtained on lysoPC/chol (1:1) vesicles which after dialysis contained only entrapped Pr3+.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol

Bilayer asymmetry in lysophosphatidylcholine/cholesterol (1:1) vesicles. A phosphorus-31 NMR study.

Prolonged sonication (3 h) of equimolar amounts of lysophosphatidylcholine (lysoPC) and cholesterol (chol) produces small unilamellar vesicles. Phosphorus-31 NMR (32.20 MHz) of the vesicles gave rise to a single peak (40.5 ppm) which was split upon addition of lanthanide ions. An additional, more intense signal appeared downfield near 51.0 ppm due to 2.4 mM Pr3+, upfield near 34.3 ppm due to 5 mM Yb3+. The more intense signals responsive to paramagnetic ions were assigned to lysoPC located in the outer vesicle leaflet; the signal not shifted by the ions was assigned to inside lysoPC. Based on peak intensities, an outside-to-inside lysoPC ratio (Ro/i) of 6.5-6.6 was determined. Essentially the same Ro/i values (6.6-6.8) were obtained when Pr3+ was present only in the vesicle interior or when Pr3+ was on the inside and Pr3+ and Yb3+ were on the outside. Ion leakage did not occur. Our data demonstrate that lysoPC/chol (1:1) vesicles are drastically asymmetric and that lysoPC shows a distinct preference for the outer bilayer leaflet.

Cholesterol

Spectroscopic investigations of the water pool in lecithin reverse micelles.

The nature of the water pool formed in the reverse micellar system, lecithin/nonpolar solvent/water, has been investigated by means of near infrared, ultraviolet, fluorescence emission and visible spectroscopic techniques. The three nonpolar solvents chosen in this study were benzene, carbon tetrachloride and cyclohexane. Near infrared spectroscopic studies revealed that the amount of water present in the bulk organic phase is negligible at all water concentrations studied in all three solvents. The results of the polarity probe and 8-anilinonaphthalene sulfonic acid (ANSA) fluorescence emission maxima studies indicate that the polarity of the water pool is much lower than that of bulk water. The difference in polarity between the water pool and bulk water decreases with increasing water concentration in benzene and carbon tetrachloride systems. However, in the cyclohexane system, at a water content of 6 moles of water per mole of lecithin, where the system is known to change from isotropic reverse micelle to anisotropic liquid crystalline state, the polarity of the water pool is found to decrease.

Anilino Naphthalenesulfonates