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Biomedical subjects

S Dey

Publications and source records attributed to S Dey.

At least 73 records · Page 4Linked to original sources
PubMed1997

Hydrofluorosis in water buffalo (Bubalus bubalis) in India.

The concentration of fluoride was determined in water, forage and urine and serum samples of buffaloes from the Unnao district of India. The water and forage samples contained 2.01 +/- 0.51 and 22.50 +/- 0.82 ppm of fluoride, respectively. The analysis of biosamples collected from the affected animals revealed higher levels of fluoride in serum (0.58 +/- 0.05 ppm) and urine (10.64 +/- 1.23 ppm). Clinical examination identified a 40.34% prevalence rate of clinical lesions suggestive of fluorosis in buffalo of this locality. Dental lesions were present invariably in all affected animals whereas lameness, painful bony exostosis and emaciation were recorded in 28.17%, 8.45% and 76.00% of the animals. Based on the clinical lesions and fluoride content in water, serum and urine, it was concluded that the problem of fluorosis in buffalo is attributable to drinking water containing toxic levels of fluoride.

Animal Feed↗
PubMed1997

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein.

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

3T3 Cells↗
PubMed1997

Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2(-1) in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2(-1) coupled to electrochemical energy.

Adenosine Triphosphatases↗
PubMed1997

Design of specific structures using alpha,beta-dehydro-phenylalanine residues: synthesis, crystal structure, and molecular conformation of Boc-L-Val-delta Phe-delta Phe-L-Val-delta Phe-delta Phe-L-Val-OCH3, a 3(10)-helical heptapeptide.

The peptide design using alpha,beta-dehydro-residues has wide applications. To design an extensive 3(10)-helical conformation, a heptapeptide Boc-L-Val-delta Phe-delta Phe-L-Val-delta Phe-delta Phe-L-Val-OCH3, with a repeat of two consecutive delta Phe residues has been synthesized using an azlactone method in solution phase. This is the first design using a repeat of two consecutive delta Phe residues. It is observed that the delta Phe in a sequence of two consecutive delta Phe residues, adopts only one set of phi, psi values, i.e., +/- 60 degrees, +/- 30 degrees, thus making it a specific design tool. The peptide crystallized from its solution in a methanol-water mixture in the space group P2(1) with a = 10.159(5)A, b = 20.057(2)A, c = 14.448(3)A, beta = 99.41(2)degrees, V = 2904(2)A3. The structure has been determined by direct methods and refined to an R value of 0.048 for 5404 observed [I > or = 3 sigma(I)] reflections. The structure consists of a heptapeptide Boc-L-Val-delta Phe-delta Phe-L-Val-delta Phe-delta Phe-L-Val-OCH3 and a solvent methanol molecule in the asymmetric unit. All peptide units in the structure are trans. As a result of six overlapping type III beta-turns formed involving seven residues and five intramolecular 4-->1 hydrogen bonds, the peptide adopts a right-handed 3(10)-helical conformation with more than two complete helical turns. It is noteworthy that starting from the Boc group to the C-terminal residue of Val, the 3(10)-helical structure is maintained well. The carbonyl oxygen atom of the Boc group is the first acceptor whereas the carbonyl oxygen atom of Val4 is the last acceptor in the helical structure of the peptide. The side chains of four delta Phe residues in this helical arrangement exist in a slightly staggered arrangement. The solvent methanol molecule interacts through its hydroxyl group and forms two intermolecular hydrogen bonds, one as a donor with a C-terminal CO group of delta Phe6 and second as an acceptor with the NH group of delta Phe2 from the N-terminal region of the peptide. Thus the solvent molecule plays a significant role in promoting a head-to-tail packing of 3(10)-helices of the peptide. There are no lateral hydrogen bonds between the helices, but there exist several van der Waals interactions involving the hydrophobic side chains of peptide molecules.

Crystallography, X-Ray↗
PubMed1997

Efflux systems and increased trypanothione levels in arsenite-resistant Leishmania.

The mechanism of resistance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mechanism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania.

ATP-Binding Cassette Transporters↗
PubMed1997

Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues.

We have measured the levels of glycosphingolipids and the activity of glycosphingolipid glycosyltransferases in human aortic intima and media from patients who died of atherosclerosis. The effects of lactosylceramide (LacCer) and glucosylceramide (GlcCer) from plaque intima on smooth muscle cell proliferation were assessed. When the GlcCer data was expressed as (micrograms GlcCer/mg cholesterol and/mg total phospholipid, a 28-fold and 7-fold increase in plaque intima compared to normal intima was observed. Similarly, the level of LacCer was elevated 5-fold and 4-fold, respectively, compared to unaffected intima. The activity of UDP-GlcCer: ceramide beta 1-->4 glucosyltransferase (GlcT-1) was similar in unaffected tissue, fatty streaks, and plaques. However, the activity of UDP-galactose: GlcCer, beta 1-->4 galactosyltransferase (GalT-2) activity was moderately higher in plaque than in unaffected tissue. LacCer, but not GlcCer derived from plaque intima exerted a approximately 2.8-fold increase in the proliferation of human aortic smooth muscle cells grown in tissue culture compared to control presumably due to a marked increase in LacCer molecular species containing C16:0, C22:1, and C24:0 fatty acids in plaque intima compared to control. In sum, our findings provide an interesting and novel pathogenic mechanism of lactosylceramide mediated plaque formation via stimulation of aortic smooth muscle cell proliferation.

Adult↗
PubMed1996

Trypanothione overproduction and resistance to antimonials and arsenicals in Leishmania.

Leishmania resistant to arsenicals and antimonials extrude arsenite. Previous results of arsenite uptake into plasma membrane-enriched vesicles suggested that the transported species is a thiol adduct of arsenite. In this paper, we demonstrate that promastigotes of arsenite-resistant Leishmania tarentolae have increased levels of intracellular thiols. High-pressure liquid chromatography of the total thiols showed that a single peak of material was elevated almost 40-fold. The major species in this peak was identified by matrix-assisted laser desorption/ionization mass spectrometry as N1,N8-bis-(glutathionyl)spermidine (trypanothione). The trypanothione adduct of arsenite was effectively transported by the As-thiol pump. No difference in pump activity was observed in wild type and mutants. A model for drug resistance is proposed in which Sb(V)/As(V)-containing compounds, including the antileishmanial drug Pentostam, are reduced intracellularly to Sb(III)/As(III), conjugated to trypanothione, and extruded by the As-thiol pump. The rate-limiting step in resistance is proposed to be formation of the metalloid-thiol pump substrates, so that increased synthesis of trypanothione produces resistance. Increased synthesis of the substrate rather than an increase in the number of pump molecules is a novel mechanism for drug resistance.

Animals↗
PubMed1996

Gene disruption of the P-glycoprotein related gene pgpa of Leishmania tarentolae.

Transfection of pgpA into Leishmania confers resistance to arsenite and antimonials. By gene targeting mediated by homologous recombination the two alleles of the pgpA gene of a L. tarentolae wild-type cell were disrupted sequentially with the neomycin and hygromycin phosphotransferase genes. This pgpA null mutant showed an increased sensitivity to arsenite and antimonite. In addition, the L. tarentolae pgpA null mutant exhibited a decreased intracellular survival inside murine macrophages. The observed phenotypes were reverted to levels not statistically different than wild-type when an intact pgpA gene was introduced into the null mutant. Disruption of the pgpA chromosomal locus in an arsenite resistant mutant indicated that PgpA is not essential for resistance to oxyanions, although it might be required in the early steps of selection when resistance is being established.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae.

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.

Animals↗
PubMed1996

Design of peptides using alpha,beta-dehydro-residues: synthesis, crystal structure and molecular conformation of Boc-L-Val-delta Phe-delta Phe-L-Val-OCH3.

The peptide Boc-L-Val-delta Phe-delta Phe-L-Val-OCH3 was synthesized by the azlactone method in solution phase, and its crystal and molecular structures were determined by x-ray diffraction method. Single crystals were grown by slow evaporation from a methanol/water solution at 6 degrees C. The crystals belong to an orthorhombic space group P212121 with a = 10.478 (6) A, b = 13.953 (I), c = 24.347 (2) and Z = 4. The structure was determined by direct methods and refined by least squares procedure to an R value of 0.052. The structure consists of a peptide and a water molecule. The peptide adopts two overlapping beta-turn conformations of Types II and I' with torsion angles: phi 1 = -54.8 (6) psi 1 = 130.5 (4), phi 2 = 65.8 (5), psi 2 = 12.8 (6), phi 3 = 79.4 (5), psi 3 = 3.9 (7) degrees. The conformation is stabilized by intramolecular hydrogen bonds involving Boc CO and NH of delta Phe3 and CO of Val1 and NH of Val4. The molecules are tightly packed in the unit cell. The crystal structure is stabilized by hydrogen bonds involving NH of delta Phe2 and CO of a symmetry related (x-1/2, 1/2-y, -z) delta Phe2. The solvent-water molecule forms two hydrogen bonds with peptide molecule involving NH of Val1 as an acceptor and another with CO of a symmetry related (1-x, y-1/2, 1/2 -z) delta Phe3 as a donor. These studies indicate that a tetrapeptide with two consecutive delta Phe residues sequenced with valines on both ends adopts two overlapping beta-turns of Types II and I'.

Crystallography↗
PubMed1996

Efflux-mediated resistance to arsenicals in arsenic-resistant and -hypersensitive Chinese hamster cells.

Several Chinese hamster V79 cell line variants resistant to arsenite and one arsenite-hypersensitive variant have been isolated. The basis for the variation in arsenite sensitivity was studied by transport experiments using radiolabeled arsenite. Two arsenite-resistant variants (As/R7 and As/R27) exhibited decreased accumulation of arsenite, and the hypersensitive variant (As/S5) exhibited increased arsenite accumulation compared with the parental line. Cells depleted of endogenous energy reserves were loaded with radiolabeled arsenite, and the rate of arsenic efflux was measured. Arsenite-resistant variants exhibited an increased rate of efflux, while the hypersensitive variant exhibited a decreased efflux rate. Efflux was decreased in cells incubated with the protonophore carbonyl cyanide m-chlorophenylhydrazine, demonstrating its energy dependence. Two inhibitors of glutathione S-transferase also decreased arsenite efflux, suggesting the involvement of an arsenite-glutathione complex. However, separation of the products of extrusion and the intracellular arsenic species by paper chromatography followed by autoradiography failed to show the appearance of an arsenite-glutathione complex in either case. Rather, all label in the product of the transport reaction appeared to be arsenite whether cells were loaded with arsenate or arsenite, indicating first that intracellular reduction of As(V) to As(III) had occurred and second that the arsenite was transported as an unconjugated species. All intracellular label was associated with high-molecular-weight material, possibly protein. Our results demonstrate the existence of an energy-dependent arsenical efflux pump in mammalian cells and show that arsenic is extruded as arsenite.

Animals↗
PubMed1996

Design of peptides: synthesis, crystal structure and molecular conformation of N-Boc-L-Val-delta Phe-L-Val-OC H3.

The dehydropeptide Boc-L-Val-delta Phe-L-Val-OC H3 was synthesized by azlactone method in solution phase. The peptide crystallized from its solution in a methanol/water mixture (70:30) in space group P2(1)2(1)2(1) with a = 13.638(1)A, b = 22.864(3)A, c = 27.600(2)A, V = 8606(1)A3. The structure was determined by direct methods and refined to an R value of 0.089 for 3326 observed [1 > or = 2 sigma(I)] reflections. The structure contains three crystallographically independent molecules. Two molecules (A and B) adopt identical conformations with phi 1(A) = -130(1), phi 1(B) = -139(1), psi 1(A) = 153(1), psi 1(B) = 145(1), phi 2(A) = 62(1), phi 2(B) = 56(1), psi 2(A) = 33(1), psi 2(B) = 33(1), phi 3(A) = -75(1), phi 3(B) = -77(1) psi 3T(A) = 152(1) and psi 3T(B) = 163(1) degrees. The conformation of the third molecule (C) is different as in that torsion angle psi 1 is rotated by 180 degrees. The backbone torsion angles are phi 1 = -128(2), psi 1 = -37(2), phi 2 = 65(1), psi 2 = 35(1), phi 3 = -84(1) and psi 3T = 169(1). It is significant that a characteristic beta-turn II conformation as observed in peptides with a delta Phe residue at (i+2) position has not been observed in this case. Thus the present structure demonstrates that an (i+2) substituted delta Phe with branched beta-carbon residue Val on both sides of it in a three peptide unit sequence does not adopt a folded conformation. The three independent molecules in the asymmetric unit form three hydrogen bonds between each pair of molecules and generates a tightly interacting unit of three molecules. These units pack into the crystalline state with hydrogen bonds involving N1, N2 and N3 of molecule C and O1' and O2' of symmetry related molecule A. The structure confirms that a peptide containing a delta Phe residue at (i+2) position with branched beta-carbon residues as its immediate neighbours on both sides does not adopt a folded conformation. This structure further demonstrates that the unfolded structures without any intramolecular hydrogen bond can be influenced by intermolecular forces, thus causing conformational variations in saturated residues. It is noteworthy that the conformation of the delta Phe residue remains unchanged in all the three molecules.

Crystallography, X-Ray↗
PubMed1996

Hyponatremia caused by a reset osmostat in a neonate with cleft lip and palate and panhypopituitarism.

A neonate with cleft lip and palate and hypopituitarism had persistent hyponatremia despite treatment with hydrocortisone, L-thyroxine, and growth hormone. Serum sodium concentration and urinary osmolality increased and decreased appropriately and concurrently with alterations in sodium and water intake. The ability to regulate serum concentrations of antidiuretic hormone at subnormal serum sodium concentrations indicated a reset osmostat.

Cleft Lip↗
PubMed1996

Design of peptides: synthesis, crystal structure and molecular conformation of N-Boc-L-Val-delta Phe-L-Ile-OCH3.

The dehydro-peptide Boc-L-Val-delta Phe-L-Ile-OCH3 was synthesized by the azlactone method in the solution phase. The peptide crystallized from a methanol/dimethyl sulfoxide (95:5) mixture in space group P6(1) with a = b = 15.312(1), c = 22.164(5) A. The structure was determined by direct methods and refined to an R value of 0.098 for 1589 observed reflections [I > or = 1.5 sigma (I)]. The peptide adopts an S-shaped conformation with torsion angles: phi 1 = -127(1), psi 1 = -44(1), phi 2 = 67(1), psi 2 = 37(1), phi 3 = -82(1) degrees. The side-chain torsion angles in delta Phe of chi 2(1) = 1(2), chi 2(2,1) = 7(2) and chi 2(2,2) = 177(1) degrees indicate that the delta Phe residue is essentially planar. In valyl residue the two side-chain torsion angles are chi 1(1) = -65(1) and chi 1(2) = 177(1), whereas the torsion angles in Ile are chi 3(1,1) = 72(2), chi 3(1,2) = -159(2), chi 3(2) = 150(2) degrees. This is the first peptide which does not adopt a folded conformation for a sequence with a delta Phe at the (i + 2) position. The molecular packing in the crystals is stabilized by several hydrogen bonds: N1-H1-...O1 = 2.77(1) A, N2-H2...O1' = 2.95(1) A, N3-H3...O2 = 2.85(1) A and a possible weak interaction N2-H2...O2' = 3.29(1) A within the columns of molecules along the c-axis and van der Waals forces between the columns.

Crystallization↗
PubMed1996

Backbone--side-chain interactions in serine. Synthesis, crystal structure and solution conformation of a linear model peptide N-Boc-L-Ser-L-Phe-OCH3.

The peptide Boc-Ser-Phe-OCH3 was synthesised by a solution-phase method using the usual workup procedure. The peptide was crystallized from a 70:30 (v/v) methanol-water mixture. The crystals are monoclinic, space group P21 with a = 5.128(2), b = 17.873(2), c = 11.386(2) A, and beta = 98.03(3) degrees. The structure was determined by direct methods and refined by structure factor least-squares procedure. The final R-value for 1499 observed reflections was 0.041. The structure contains one peptide and one solvent water molecule. The peptide adopts a beta-strand-like conformation with phi 1 = -100.3(5), psi 1 = 99.9(5), phi 2 = -122.2(5), psi T2 = -172.5(6) degrees. The Ser side-chain assumes an extended conformation with chi 11 = -177.0(4) degrees. The O gamma H group of serine acts as a proton donor in an intramolecular weak hydrogen bond with (Ser) O'1 [O gamma 1 - H gamma 1 ... O'1 = 3.253(6) A]. The Phe side-chain adopts a staggered conformation with chi 1(2) = -70.9(6), chi 2,1 (2) = 88.4(7) degrees, chi 2,2(2) = -89.2(6) degrees. The water molecule generates a loop through two hydrogen bonds with O gamma 1 [OW ... O gamma 1 = 2.893(5) A] and O'2 [OW ... O'2 = 2.962(7) A] atoms. The unit-translated peptide molecules along the a-axis are held by hydrogen bonds: N1 - H1 ... O2 (chi - 1, y, z) = 2.954(4) A and N2 - H2 ... O'1 (chi + 1, y, z) = 2.897(6) A in a manner similar to those observed in parallel beta-pleated sheet structures. There is an additional interaction involving O gamma 1 and the water molecule [OW ... O gamma 1 (chi + 1, y, z) = 2.789 (4) A]. The strong NOE peak of Ci(H) ... Ni + 1 (H) and a simultaneous weak NOE peak of Ni(H) ... Ni + 1 (H) in the ROESY spectra of two-dimensional NMR in dimethyl sulfoxide indicate a beta-strand-like conformation for the peptide in solution.

Models, Molecular↗