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D Singh

Publications and source records attributed to D Singh.

At least 19 recordsLinked to original sources
PubMed1992

Behaviour of a glycosphingolipid with unsaturated fatty acid in phosphatidylcholine bilayers.

N-(Oleoyl)galactosylceramide with perdeuterated acyl chain was prepared by partial synthesis, and studied by wide line 2H-NMR in phospholipid liposomes. Spectra were obtained for low glycolipid concentrations in bilayers of dimyristoyl-, distearoyl-, and 1-palmitoyl-2-oleoylphosphatidylcholines. In an attempt to isolate the effects of glycosphingolipid fatty acid cis unsaturation on glycolipid behaviour in membranes, spectral findings related to the above species were compared to literature NMR data for pure 1-palmitoyl-2-oleoylphosphatidylcholine bilayers in which the oleoyl chain of the phospholipid had been deuterated, and to analogously deuterated glycerol based lipids in Acholeplasma laidlawii membranes. The results for N-(oleoyl-d33)galactosylceramide proved to be qualitatively and quantitatively very similar to published data dealing with glycerol based lipids at comparable temperatures. In addition, the results were strikingly similar for glycolipids dispersed in saturated and unsaturated phospholipid host matrices. It would appear that the primary effects of cis 9,10 fatty acid unsaturation in glycosphingolipids (at low concentration in fluid phospholipid membranes) are the same as those of fatty acid cis unsaturation in glycerolipids. It further appears that the overall dynamic behaviour of N-(oleoyl)galactosylceramide in fluid phospholipid membranes is very similar to that of glycerolipids with comparable acyl chains.

Fatty Acids, Unsaturated↗
PubMed1992

Glycosphingolipid phase behaviour in unsaturated phosphatidylcholine bilayers: a 2H-NMR study.

2H-NMR was employed to consider the arrangement of a glycosphingolipid, N-(lignoceroyl-d47)galactosylceramide, in bilayers of the mono-unsaturated phospholipid, 1-stearoyl-2-oleoylphosphatidylcholine. The deuterated glycolipid prepared by partial synthesis was incorporated at concentrations ranging from 5 mol% to 53 mol% into unsonicated liposomes, and its spectra were recorded from +76 degrees C to -10 degrees C. First spectral moments were plotted as a function of temperature for each sample composition and, along with inspection of the spectra, were employed to infer a phase diagram describing glycolipid behaviour in the unsaturated phospholipid host matrix. It was possible to refine the result using 2H-NMR difference spectroscopy. The phase diagram obtained was indicative of peritectic behaviour. At glycolipid concentrations exceeding about 20 mol% there was considerable tendency to glycolipid phase separation--as indicated by coexistence of fluid phospholipid-enriched and gel phase glycolipid-enriched domains over a wide range of temperatures, and by coexistence of distinct ordered phase domains at lower temperature. In contrast, at lower glycolipid concentrations reflective of many biological membranes, the lipid components were miscible in both the liquid crystal and gel phases, with only a narrow temperature range of fluid and ordered phase coexistence. For the fluid phase at low glycolipid concentrations, spectra of the deuterated glycolipid 24-carbon fatty acid suggest that orientational order is low for a number of methylene groups near the methyl end of the chain.

Deuterium↗
PubMed1992

Glycosphingolipids: 2H NMR study of the influence of ceramide fatty acid characteristics on the carbohydrate headgroup in phospholipid bilayers.

Galactosylceramides bearing a variety of different pure fatty acid chains were 2H labeled in the carbohydrate headgroup at C6 of the terminal galactose residue, for study by 2H NMR. Fatty acids investigated included the 24-carbon saturated lignoceric acid, 18-carbon saturated stearic acid, cis-9,10-unsaturated oleic acid, and D- and L-stereoisomers of alpha-hydroxystearic acid. Headgroup-deuterated glycolipids were incorporated at 10 mol % into unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine, and 2H NMR spectra were recorded at 65 and 40 degrees C. Under these experimental conditions, the membranes studied were primarily in the liquid-crystalline phase. At a given temperature, spectra for deuterated galactosylceramides dispersed in the fluid phase were remarkably similar, regardless of the nature of the fatty acid attached to the glycolipid sphingosine backbone. In each case, the spectrum consisted of a superposition of two quadrupolar powder patterns of approximately equal intensity. The spectra may be interpreted as arising from equal populations of two stereoisomers (pro-R and pro-S) of the deuterated galactose hydroxymethyl function, which is undergoing rapid (greater than 10(6) s-1) interconversion among the possible rotamers about the C5-C6 bond of the sugar ring. Within experimental error, the only fatty-acid-induced spectral difference detected among these glycosphingolipids deuterated in the carbohydrate headgroup was in the species with alpha-hydroxy-substituted fatty acids. At 65 degrees C, N-(D-alpha-hydroxy)stearoyl- and N-(L-alpha-hydroxy)stearoylgalactosylceramide gave rise to the same quadrupole splittings, but these differed marginally from the splittings observed for the other glycolipids studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty Acids↗
PubMed1992

Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II.

The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of the expressed protein. Control and experimental pEX-1-Na+/H+ exchanger protein was purified on a p-aminophenyl beta-D-thiogalactopyranoside-agarose column. Purified Ca2+/calmodulin-dependent protein kinase II readily phosphorylated the Na+/H+ exchanger protein in a Ca(2+)- and calmodulin-dependent manner in vitro, but this region of the protein was not a substrate for purified protein kinase C or for the catalytic subunit of cyclic AMP-dependent protein kinase. Control-expressed beta-galactosidase was phosphorylated to a maximal level of 0.77 +/- 0.17 mol of Pi/mol (mean +/- S.E.M., n = 6) whereas the fusion protein was phosphorylated to a maximal level of 4.09 +/- 0.39 mol of Pi/mol (n = 6), suggesting one site of phosphorylation in beta-galactosidase and three in the C-terminal domain of the Na+/H+ exchanger. Examination of the deduced amino acid sequence of this part of the exchanger reveals three consensus sequences for Ca2+/calmodulin-dependent protein kinase II. These results suggest that the exchanger may be directly regulated in vivo by calmodulin-dependent protein kinase II but not by protein kinase C or cyclic AMP-dependent protein kinase.

Amino Acid Sequence↗
PubMed1992

Effects of fatty acid alpha-hydroxylation on glycosphingolipid properties in phosphatidylcholine bilayers.

The role of glycosphingolipid fatty acid alpha-hydroxylation as a modulator of glycolipid organization and dynamics was considered by 2H-NMR in bilayer membranes. For these experiments, galactosylceramides were prepared in which the natural fatty acid mixture was replaced with perdeuterated 18-carbon hydroxylated or non-hydroxylated stearic acid. The L-stereoisomer of N-(alpha-OH-stearoyl-d34)galactosylceramide and its naturally-occurring D-alpha-OH analogue, were isolated for independent study. Bilayers were formed using 10 mol% galactosylceramide in a shorter chain phospholipid, dimyristoylphosphatidylcholine, in an attempt to reproduce several features of glycolipid-phospholipid interactions typical of cell membranes. Spectra of deuterated galactosylceramide in gel phase phospholipid membranes indicated that alpha-hydroxylation led to greater motional freedom and/or conformational disorder, with no measurable difference between D- and L-alpha-OH fatty acid derivatives. In fluid phosphatidylcholine bilayers the effects were modest. Glycolipid fatty acid hydroxylation led to broadening of the range of order parameters associated with methylene groups near the membrane surface (frequently referred to as the 'plateau region') - this effect being more marked for the naturally-occurring (D) stereoisomer. The degree of overall molecular order sensed by the glycolipid fatty acid chain in a fluid host matrix was minimally affected by alpha-hydroxylation; although the plateau region of the D isomer was slightly more ordered than that of the L isomer and the non-hydroxylated species. These results suggest that a significant aspect of the alpha-hydroxy group effect on glycosphingolipid behaviour in bilayer membranes with low glycolipid content was interference with glycolipid packing amongst host phospholipids in the upper portion of the acyl chains. For the D stereoisomer, there was some evidence that the hydroxy group led to strengthening of interlipid interaction near the membrane surface.

Fatty Acids↗
PubMed1992

Oligosaccharide order in a membrane-incorporated complex glycosphingolipid.

Galactosylceramide (GalCer) and the ganglioside, GM1, were 2H-labelled at C-6 (the hydroxymethyl moiety) of their single terminal galactosyl residues. Each deuterated glycosphingolipid was incorporated at a biologically relevant low concentration into multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). 2H-NMR spectra of aqueous dispersions of GalCer-POPC in the liquid crystal phase were characteristic of restricted headgroup motion (ordering) with effective axial symmetry. The degree of headgroup ordering was analogous to that of GalCer in pure aqueous multibilayers (Skarjune, R. and Oldfield, E. (1979) Biochim. Biophys. Acta 556, 208-218). In the case of GM1, 2H-labelled in the terminal galactose residue of the pentasaccharide headgroup, the 2H-NMR spectra were remarkably like those of the simple glycolipid, GalCer. This suggests substantial restriction of motion about the glycosidic and sugar-ceramide bonds of the complex GM1 headgroup, and that both lipids have comparable degrees of orientational averaging (fluctuation) about the bilayer normal. The result is the first direct demonstration that headgroup orientational order can exist for a complex glycolipid incorporated into 'fluid' bilayer membranes. Such behaviour argues for the possibility of modulation of membrane receptor properties through surface effects on average headgroup orientation and conformation.

Animals↗
PubMed1992

Phase II trial of 5-fluorouracil, high-dose leucovorin calcium, and dipyridamole in advanced prostate cancer.

To examine the effect of altering intracellular folate pools on the efficacy of 5-fluorouracil (FUra) in the treatment of advanced prostate cancer, we performed a phase II trial of FUra (300-370 mg m-2 day-1 x 5 as an i.v. bolus) combined with high-dose folinic acid (500 mg m-2 day-1 x 5.5 days by continuous i.v. infusion) and dipyridamole (75 mg p.o. every 6 h x 5.5 days) administered on a 28-day schedule in patients with stage D2 disease. A group of 13 patients have been treated. The median age was 68 years (range 48-78 years); the performance status ranged from 50% to 90%. Among 12 evaluable patients, there were no objective responders; the median time to progression was 1.9 months. Median survival after entry on this trial was 8.6 months. Treatment with FUra, high-dose folinic acid and dipyridamole was well tolerated. Only one episode each of grade 3 leukopenia, granulocytopenia, and thrombocytopenia was observed. These results suggest that, despite previous trials demonstrating activity for FUra in stage D2 prostate cancer, this disease may be relatively resistant to fluoropyrimidines and, thus, less amenable to biochemical modulation with high-dose folinic acid and dipyridamole.

Aged↗
PubMed1992

Dental caries and periodontal disease in Fiji.

A national oral survey was conducted in Fiji in 1985/86. The results showed that the prevalence of caries was generally low and most commonly affected pit and fissure surfaces. Comparisons with previous surveys in 1948 and 1965 showed that the prevalence had fallen in primary teeth but remained relatively constant in permanent teeth. There were significant variations in prevalence between residential locations but not between ethnic groups. Periodontal disease was found to be a major public health problem. Except for the percentage of young children with caries-free dentitions, the current status of oral health in Fiji was generally better than that proposed in the WHO goals for the year 2000. The training of dental hygienists to help in the prevention of both dental caries and periodontal disease is discussed.

Adolescent↗
PubMed1991

Seed protein fractions and amino acid composition in gram (Cicer arietinum).

Six chickpea strains were analysed for their protein content and various protein fractions. The protein content ranged from 20.9-25.27%. Albumin, globulin, prolamin and glutelin contents ranged from 8.39-12.31%; 53.44-60.29%; 3.12-6.89% and 19.38-24.40% respectively. Salt soluble proteins (albumin + globulin) and globulins resolved into 19-23 bands whereas albumin proteins resolved into 30-34 bands. The molecular weights of various polypeptides ranged from 10-91 kD. Amino acid analysis of total proteins revealed that glutamic acid was present in maximum concentration followed by aspartic acid and arginine. Just like other pulse proteins, chick pea proteins were also found deficient in sulphur containing amino acids.

Albumins↗
PubMed1991

Measurement of neonatal skinfold thickness--is it of any clinical relevance?

The skinfold thickness (SFT) was measured in 750 Punjabi newborns at triceps and subscapular sites using a Harpenden's Caliper. It was correlated with various maternal and neonatal factors. SFT increased with increasing gestation but showed a decline after 40 weeks. There was a positive correlation of SFT with birth weight and length of the baby in both sexes. The correlation co-efficient for all these parameters was 0.9. The female babies had a higher SFT at all weight and length groups. Increasing maternal age, parity, weight and height all influenced the neonatal SFT positively. Mothers with higher SFT produced babies with more skinfold thickness. Similar relationship was observed between birth weight and these maternal factors. While severe pre-eclampsia and eclampsia led to a significant fall in SFT, hypertension alone did not affect it. A higher than normal SFT was seen among infants of diabetic mothers. It was concluded that the SFT does not give any additional information than that provided by the commonly measured parameters like birth weight and length.

Adult↗
PubMed1991

Hydatid disease.

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Child↗
PubMed1990

Oligonucleotides, part 5+: synthesis and fluorescence studies of DNA oligomers d(AT)5 containing adenines covalently linked at C-8 with dansyl fluorophore.

The synthesis of oligodeoxynucleotides d(AT)5 in which specific adenines are linked at C-8 position with dansyl fluorophores via a variable polymethylene spacer chain are reported. This was achieved by a strategy involving prelabelling at the monomeric stage followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled oligonucleotides. Several mono and polydansyl d(AT)5 derivatives in which the fluorophore is linked via ethylene, tetramethylene and hexamethylene spacer arms were synthesised for a systematic study of their fluorescence characteristics. It was observed that (i) enhancements in fluorescence intensity and emission quantum yields are seen due to multiple labelling, (ii) the magnitude of enhancements are related to labelling configuration and (iii) quenching efficiency is minimal with shorter and rigid spacer arms. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.

Adenine↗
PubMed1990

Point mutations define positions in HLA-DR3 molecules that affect antigen presentation.

Allelic differences in major histocompatibility complex (MHC)-encoded class II molecules affect both the binding of immunogenic peptides to class II molecules and the recognition of MHC molecule-peptide complexes by T cells. As yet, there has been no extensive mapping of these functions to the fine structure of human class II molecules. To determine sites on the HLA-DR3 molecule involved in antigen presentation to T cells, we used monoclonal antibodies specific for HLA-DR3 to immunoselect mutants of a B-lymphoblastoid line. We located the sites of single amino acid substitutions in the HLA-DR3 molecule and correlated these structural changes with patterns of recognition by HLA-DR3-restricted, antigen-specific T cells, allospecific T cells, and allospecific anti-DR3 monoclonal antibodies. We analyzed seven mutations. One mutation, at position 74 in domain 1 of the DR beta chain, affected recognition by all T cells tested, whereas others, at positions 9, 45, 73, 151, and 204 of the DR beta chain and position 115 of the DR alpha chain, altered recognition by some T cells, but not others. Each of the substitutions resulted in a unique pattern of T-cell stimulation. In addition, each T-cell clone recognized a different subset of the mutants. These results indicate that different residues of the DR3 molecule are involved in presentation of antigen to different DR3-restricted T cells. These studies further show that substitutions which most likely affect peptide binding alter recognition of DR3 molecules by an alloreactive T-cell clone and some allospecific antibodies.

Alleles↗