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Biomedical subjects

R Sharma

Publications and source records attributed to R Sharma.

At least 91 records · Page 5Linked to original sources
PubMed2002

Performance of an age series of Alnus-cardamom plantations in the Sikkim Himalaya: nutrient dynamics.

Nutrient cycling, nutrient use efficiency and nitrogen fixation in an age series of Alnus-cardamom plantations were studied in the eastern Himalaya. The impact of stand age (5, 10, 15, 20, 30 and 40 years) on the nutrient dynamics of mixtures of N2-fixing (Alnus nepalensis) and non-N2-fixing (large cardamom) plants was assessed. Foliar nutrient concentrations of Alnus decreased with advancing age groups of plantations and showed an inverse relationship with stand age. Annual N fixation increased from the 5-year-old stand (52 kg ha(-1)), peaking in the 15-year-old stand (155 kg ha(-1)) and then decreased with increasing plantation age. Nitrogen and phosphorus uptake was lowest in the 40-year-old stand, and highest in the 15- and 5-year-old stand, respectively. Nutrient storage in understorey cardamom was very high: up to 31 % N and 59 % P of the stand total in the 15-year-old stand. Nutrient use efficiency was higher (with faster turnover times) in younger stands and decreased (with slower turnover times) in older plantations. Nitrogen retranslocation showed a strong positive relationship with stand age, while that of P was inversely related to stand age. Nutrient standing stock, uptake and return were also highest in the 15-year-old stand. Nitrogen and P cycling in Alnus-cardamom plantations was functionally balanced. Nutrient cycling and dynamics indicated that Alnus-cardamom plantations performed sustainably up to 15-20 years. The management practice should be altered to incorporate replantation after this age.

Algorithms↗
PubMed2002

Construction of an RAPD linkage map and localization of QTLs for oleic acid level using recombinant inbreds in mustard (Brassica juncea).

RAPD markers were employed for construction of a linkage map and localization of QTLs for oleic acid level using a set of 94 recombinant inbred lines (RILs) of mustard (Brassica juncea L.) as a mapping population. Only 30% of the 235 random primers used were useful in terms of polymorphism detected and the reproducibility of those patterns. Normal Mendelian segregation was observed for the majority of the 130 markers obtained with 71 informative primers; only 13.1% deviated (P < 0.01) from the expected 1:1 ratio. One-hundred and fourteen markers were assigned to 21 linkage groups (LGs) covering a total length of 790.4 cM with an average distance of 6.93 cM between markers. Two quantitative trait loci (QTL) for oleic acid level were mapped to 14- and 10.6-cM marker intervals on two different LGs. Both loci together explained 32.2% of phenotypic variance. One major QTL explained 28.5% of the trait variance observed in this species.

Breeding↗
PubMed2002

Non-obese hyperlipidemic Asian northern Indian males have adverse anthropometric profile.

BACKGROUND AND AIMS: Hyperlipidemia is commonly ascribed to obesity. We studied the association of anthropometric profile and nutrient intake with hyperlipidemia in non-obese Asian Indian males. METHODS AND RESULTS: In a case-control study, non-diabetic, non-obese males with body mass index (BMI) < 25 Kg/m2 having primary hyperlipidemia (n = 50) were compared with healthy normolipidemic males (BMI < 25 Kg/m2, n = 50). In this study, BMI (p < 0.001), waist circumference (WC) (p < 0.001), waist/hip ratio (p < 0.01), skinfolds (p < 0.001), sum of four skinfolds (p < 0.001), and percentage of body fat (%BF) (p < 0.001) were significantly higher in hyperlipidemic subjects as compared to normolipidemic controls. High %BF (> 25%) was observed in 26% of hyperlipidemic subjects and in 8% normolipidemic controls (p < 0.01). Using analysis of covariance (after adjusting for BMI), WC (p < 0.001), %BF (p < 0.01) and %BF/BMI ratio (p < 0.01) were significantly higher in the hyperlipidemic subjects. Intakes of total calories, total fat, saturated fat, carbohydrates, n-3 and n-6 fatty acids, dietary fiber, vitamin E and n-6/n-3 fatty acids ratio were statistically comparable between the two groups. In hyperlipidemic subjects biceps skinfold correlated negatively to levels of high-density lipoprotein cholesterol (r = -0.28, p < 0.05) and serum triglycerides correlated positively to intakes of carbohydrate (r = 0.31, p < 0.05), and dietary n-3 fatty acids (r = 0.34, p < 0.05). CONCLUSIONS: Hyperlipidemic Asian Indian males, defined as "non-obese" based on BMI, had adverse profile of anthropometric parameters and excess %BF as compared to normolipidemic males. Therefore, while dealing with hyperlipidemic Asian Indians, physicians should consider anthropometric parameters (WC), %BF, and %BF/BMI ratio in addition to BMI in the clinical assessment.

Adipose Tissue↗
PubMed2002

Experimental studies on waste paper pulp biodegradation.

In continuation of their studies on Biodegradation of cellulosic waste materials, the authors have presented their experimental data on biodegradation on waste paper pulp. Greater efficiency in the cow dung and M3 culture experimental setups have been pointed out and discussed. Necessity of detailed data evaluation of temperature, pH, dry weight, cellulose, protein, carbohydrate, Nitrate, Phosphate & Potassium levels is suggested. Derivation of %, absolute and increase/decrease % of BOO, COD and VS have been pointed out for the first time in solid waste Biodegradation. Further data evaluation and derivation of critical reaction rate kinetics have been recommended for further studies in solid waste-degradation to achieve quicker Bioconversion of cellulosic wastes into compost.

Biodegradation, Environmental↗
PubMed2001

Hyperactive phosphoinositide signaling pathway in platelets of depressed patients: effect of desipramine treatment.

There is some evidence to suggest that certain neurotransmitter receptors, such as adrenergic and serotonergic receptors and receptor-linked signaling systems, may be altered in depression. Serotonin(2A) and alpha(2)-adrenergic receptors are linked to the phosphoinositide (PI) signaling system in platelets and brain. To examine if the PI signaling system is altered in depression, we studied thrombin- and sodium fluoride-stimulated inositol phosphate(1) (IP(1)) formation before and during desipramine (DMI) treatment in platelets of depressed patients and normal control subjects. We determined thrombin- and sodium fluoride-stimulated IP(1) formation in platelets obtained from hospitalized depressed patients during a drug-free baseline period and after 6 weeks of DMI treatment, and drug-free non-hospitalized normal control subjects. Depressed subjects were diagnosed according to DSM-IV criteria, and severity of illness was assessed with the Hamilton Depression Rating Scale. We observed that thrombin-stimulated IP(1) formation in platelets of depressed patients was significantly higher compared with that of normal control subjects. There were no significant differences in sodium fluoride-stimulated IP(1) formation between depressed patients and normal control subjects. We also did not find any significant effect of treatment with DMI on either thrombin- or sodium fluoride-stimulated IP(1) formation in platelets of depressed patients, which continued to be significantly higher after 6 weeks of treatment with DMI, compared with normal control values. Our studies found a hyperactive PI signaling system in platelets of depressed patients. This hyperactive system may be related either to an increased number of thrombin receptors or to a generalized overstimulation of this pathway; however, since we did not observe any differences in sodium fluoride-stimulated IP(1) formation, it appears that, although the sites distal to the receptors may be altered, this abnormality is probably not related to the abnormalities in G proteins.

Adult↗
PubMed2001

Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress.

To explore the role of lipid peroxidation (LPO) products in the initial phase of stress mediated signaling, we studied the effect of mild, transient oxidative or heat stress on parameters that regulate the cellular concentration of 4-hydroxynonenal (4-HNE). When K562 cells were exposed to mild heat shock (42 degrees C, 30 min) or oxidative stress (50 microM H2O2, 20 min) and allowed to recover for 2 h, there was a severalfold induction of hGST5.8, which catalyzes the formation of glutathione-4-HNE conjugate (GS-HNE), and RLIP76, which mediates the transport of GS-HNE from cells (Awasthi, S., Cheng, J., Singhal, S. S., Saini, M. K., Pandya, U., Pikula, S., Bandorowicz-Pikula, J., Singh, S. V., Zimniak, P., and Awasthi, Y. C. (2000) Biochemistry 39, 9327-9334). Enhanced LPO was observed in stressed cells, but the major antioxidant enzymes and HSP70 remained unaffected. The stressed cells showed higher GS-HNE-conjugating activity and increased efflux of GS-HNE. Stress-pre-conditioned cells with induced hGST5.8 and RLIP76 acquired resistance to 4-HNE and H2O2-mediated apoptosis by suppressing a sustained activation of c-Jun N-terminal kinase and caspase 3. The protective effect of stress pre-conditioning against apoptosis was abrogated by coating the cells with anti-RLIP76 IgG, which inhibited the efflux of GS-HNE from cells, indicating that the cells acquired resistance to apoptosis by metabolizing and excluding 4-HNE at a higher rate. Induction of hGST5.8 and RLIP76 by mild, transient stress and the resulting resistance of stress-pre-conditioned cells to apoptosis appears to be a general phenomenon since it was not limited to K562 cells but was also evident in lung cancer cells, H-69, H-226, human leukemia cells, HL-60, and human retinal pigmented epithelial cells. These results strongly suggest a role of LPO products, particularly 4-HNE, in the initial phase of stress mediated signaling.

ATP-Binding Cassette Transporters↗
PubMed2001

Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis.

Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system.

Adenosine Triphosphate↗
PubMed2001

High tumour necrosis factor-alpha levels are associated with exercise intolerance and neurohormonal activation in chronic heart failure patients.

Immune activation plays an important role in the progression of chronic heart failure (CHF). We sought to investigate whether different degrees of tumor necrosis factor-alpha (TNF-alpha) activation are associated with exercise intolerance, neurohormonal activation and alterations in muscle mass and function in patients with CHF without cardiac cachexia. Patients were divided into quartiles according to their TNF levels (first quartile: 0.98-4.90 pg/ml, second quartile: 5.00-6.60 pg/ml; third quartile 6.80-9.00 pg/ml; fourth quartile 9.80-32.00 pg/ml). Patients underwent cardiopulmonary exercise testing, quadriceps muscle strength test, quadriceps fatigue test, and assessment of thigh muscle and fat cross-sectional area (CSA) by computerized tomography scanning. Patients in the highest TNF quartile had the lowest peak oxygen consumption [13.1 (+/-4.1) ml/kg/min vs 18.1 (+/-5.3), 18.8 (+/-4.8) and 18.7 (+/-5.6) ml/kg/min, P<0.01] the greatest relation of ventilation and dioxide production (VE/VCO(2)) slope (P<0.05) and the most elevated catecholamine levels (P<0.05) compared to patients in the first three quartiles. Patients with the lowest TNF levels had preserved thigh muscle size and quadriceps strength. Strength/muscle CSA was similar in the four groups. Muscle strength during fatigue testing was significantly lower in the fourth quartile (P=0.01) compared with the other three groups. In CHF patients only the highest levels of TNF are associated with poor functional status and neurohormonal activation. This group of patients may represent the appropriate target population for TNF antagonism.

Aged↗
PubMed2001

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Caco-2 Cells↗
PubMed2001

RLIP76 is the major ATP-dependent transporter of glutathione-conjugates and doxorubicin in human erythrocytes.

We have recently demonstrated that RLIP76, a Ral-binding GTPase activating protein mediates ATP-dependent transport of glutathione (GSH) conjugates of electrophiles (GS-E) as well as doxorubicin (DOX), and that it is identical with DNP-SG ATPase, a GS-E transporter previously characterized by us in erythrocyte membranes (Awasthi et al. Biochemistry 39, 9327-9334). Multidrug resistance-associated protein (MRP1) belonging to the family of the ABC-transporters has also been suggested to be a GS-E transporter in human erythrocytes. Using immunological approaches, the present studies were designed to elucidate the relative contributions of RLIP76, MRP1, and P-glycoprotein (Pgp), in the ATP-dependent transport of GS-E and DOX in human erythrocytes. In Western blot analyses using antibodies against RLIP76, a strong expression of RLIP76 was observed in erythrocytes. Immunohistochemical studies using a fluorescent probe showed association of RLIP76 with erythrocyte membrane, which was consistent with its transport function. Neither MRP1 nor Pgp were detected in erythrocytes when the antibodies against MRP1 or Pgp were used. In erythrocyte inside-out vesicles (IOVs) coated with antibodies against RLIP76, a dose-dependent inhibition of the ATP-dependent transport of DOX and GS-E, including S-(dinitrophenyl)glutathione (DNP-SG), leukotriene C(4), and the GSH conjugate of 4-hydroxynonenal, was observed with a maximal inhibition of about 70%. On the contrary, in the IOVs coated with the antibodies against MRP1 or Pgp no significant inhibition of the ATP-dependent transport of these compounds was observed. These findings suggest that RLIP76 is the major ATP-dependent transporter of GS-E and DOX in human erythrocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

The cysteine-rich amino-terminal domain of ZntA, a Pb(II)/Zn(II)/Cd(II)-translocating ATPase from Escherichia coli, is not essential for its function.

Soft metal-translocating P1-type ATPases have a distinctive amino-terminal domain that contains one to six copies of the conserved metal-binding motif, GXXCXXC. ZntA from Escherichia coli, a Pb(II)-, Zn(II)-, and Cd(II)-transporting ATPase, has an approximately 120 residue amino-terminal domain with one copy of the GXXCXXC motif as well as four additional cysteine residues. The function of this domain was investigated by constructing a mutant of ZntA lacking the first approximately 100 residues. The mutant, DeltaN-ZntA, was able to confer resistance to Pb(II), Zn(II), and Cd(II) salts, in a manner similar to ZntA. The soft metal dependent ATP hydrolysis activity of purified DeltaN-ZntA was characterized. Purified DeltaN-ZntA and ZntA were both inactivated by oxidation. The K(m) for MgATP was unchanged for DeltaN-ZntA relative to ZntA. DeltaN-ZntA displayed the same metal ion specificity as ZntA. Thiolates increased the activities of both ZntA and DeltaN-ZntA. The V(max) values for DeltaN-ZntA were approximately 3-fold lower than for ZntA for all three metal ions. Thus, the amino-terminal domain is not essential for the function of ZntA or for conferring specificity toward particular soft metals. Its function may be to increase the overall catalytic rate by increasing the rate of metal ion binding to the transporter. Residues involved in the ATP-dependent soft metal ion-translocating mechanism as well as those responsible for recognition of specific metal ions must be part of the core structure of the P1-type ATPases.

Adenosine Triphosphatases↗
PubMed2001

Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase.

In humans, beta-hexosaminidase A (alphabeta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta-hexosaminidase B (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from positive in alpha to negative in beta is consistent with the lower K(m) of hexosaminidase S, and the much higher K(m) and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys hexosaminidase S detected little change in V(max) but a 2-fold increase in K(m) for the sulfated substrate. Its K(m) for the nonsulfated substrate was unaffected. When alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) by substituting Gln produced a hexosaminidase S with a K(m) decrease of 3-fold and a V(max) increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K(m) was increased 9-fold and its V(max) decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its betaAsp(453) becomes protonated.

Amino Acid Sequence↗
PubMed2001

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences.

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.

ATP-Binding Cassette Transporters↗