Structural, magnetic, and heat-capacity studies on Zn- and Ga-substituted PrBa2Cu3O7- delta.
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Journal Article↗
Biomedical subjects
R Prasad
Publications and source records attributed to R Prasad.
Cadmium-induced nephrotoxicity in rhesus monkeys (Macaca mulatta) in relation to protein calorie malnutrition.
In this study, we compared results obtained in protein calorie malnourished (PCM) monkeys and controls given Cd2+ (5 mg Cd2+/kg body wt./day) orally for 24 weeks. After 16 weeks of Cd2+ exposure, an indolent renal failure develops in PCM monkeys which resulted in significant increase in urinary excretion of total protein, Cd2+, Zn2+ and Ca2+ as compared to corresponding to Cd(2+)-treated control group. In isolated proximal tubule brush border membrane vesicles (BBMV), Cd2+, Zn2+ and Ca2+ transport were significantly impaired in Cd(2+)-exposed PCM monkeys as compared to Cd(2+)-treated controls. The mechanism of higher urinary excretion of Cd2+, Zn2+ and Ca2+ was examined by analyzing the kinetic parameters of transport systems. The kinetic studies of Cd2+, Zn2+ and Ca2+ transport systems in the BBMV preparations of Cd(2+)-exposed PCM monkeys exhibited a significant decrease in Vmax and an appreciable increase in Km as compared to Cd(2+)-treated controls. These findings suggested that Cd2+ treatment of PCM monkeys caused either a decrease in the number of transporters in the brush border membrane or an increase in the number of less active transporters for Cd2+, Zn2+ and Ca2+. Furthermore, brush border membrane-bound enzymes, viz. alkaline phosphatase and leucine aminopeptidase, activities were significantly impaired in Cd(2+)-exposed PCM monkeys. Cadmium content in kidney cortex of Cd(2+)-exposed PCM monkeys was 3.34-fold higher than Cd(2+)-exposed controls. These findings also established that Cd2+ not bound to metallothionein (MT) was significantly higher in Cd-exposed PCM monkeys, which may be an important determinant in renal toxicity by interacting with sensitive sites in the renal cells and causing renal damage in Cd-exposed PCM monkeys.
Administration, Oral↗
Expression of the mcrA gene of Escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region.
The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA+/RglA+ phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA+/RglA- phenotype and directed production of a 23-kDa polypeptide. Smaller inserts displayed McrA-/RglA- phenotypes, though a 0.7-kb insert did direct production of a 24-kDa polypeptide. A construct carrying the 1.0-kb mcrA insert yielded a single 1.3-kb transcript. The mcrA transcript was found to start from C, G, T and G, namely the fourth, fifth, sixth and seventh nucleotides (nt), respectively, downstream from the last nt of the putative -10 region. Two mcrA transcriptional/transational fusions were made in the pT7-7 expression vector and the protein encoded by these constructs were analysed. Regulation of mcrA expression was studied by quantitative Northern analysis of RNA from various mcrA clones. Together with a computer analysis of the translation initiation region in these mRNAs, the results suggest that the expression of mcrA may be regulated at the translational level.
Bacterial Proteins↗
Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.
DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.
Base Sequence↗
DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract.
The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.
Animals↗
Molecular cloning and characterization of a novel gene of Candida albicans, CDR1, conferring multiple resistance to drugs and antifungals.
By functional complementation of a PDR5 null mutant of Saccharomyces cerevisiae, we have cloned and sequenced the multidrug-resistance gene CDR1 of Candida albicans. Transformation by CDR1 of a PDR5-disrupted host hypersensitive to cycloheximide and chloramphenicol resulted in resistance to cycloheximide, chloramphenicol and other drugs, such as the antifungal miconazole, with collateral hypersensitivity to oligomycin, nystatin and 2,4 dinitrophenol. Our results also demonstrate the presence of several PDR5 complementing genes in C. albicans, displaying multidrug-resistance patterns different from PDR5 and CDR1. The nucleotide sequence of CDR1 revealed that, like PDR5, it encodes a putative membrane pump belonging to the ABC (ATP-binding cassette) superfamily. CDR1 encodes a 1501-residue protein of 169.9 kDa whose predicted structural organization is characterized by two homologous halves, each comprising a hydrophobic region with a set of six transmembrane stretches, preceded by a hydrophilic nucleotide binding fold.
Amino Acid Sequence↗
Thyroid function during a prolonged stay in Antarctica.
Adaptation of the thyroid gland to the Antarctic environment was studied in nine healthy euthyroid tropical men of the Sixth Indian Antarctic Expedition during 1 year of their residence at polar latitudes. Circulatory concentrations of thyroid hormones, total T4 (TT4), total T3 (TT3), free T4 (FT4), free T3 (FT3), reverse T3 (rT3), thyroxine binding globulin (TBG), T3 uptake and thyroid stimulating hormone (TSH) were estimated in New Delhi and during the first week of each month of the stay in Antarctica. At the end of the Austral summer in March, the TT3 concentrations were found to be significantly lower (P < 0.01) compared to values recorded in New Delhi and showed a significant increase (P < 0.05) during the Austral winter in August. The mean TT3 concentrations from May to December were found to be significantly higher than the March or April values. Plasma TT4 and rT3 concentrations tended to decline in March but remained unaltered during the entire period in Antarctica. The FT4, FT3, TBG and T3 uptake did not show any appreciable change. Though, the TT3:TT4 ratio tended to decline in March and April suggesting decreased peripheral conversion of T4 to T3 as the possible mechanism for a decline in TT3 in March. physical exertion and prolonged exposure to extreme cold appeared to be the major contributory factors. The TSH concentration in March, April, November and December were found to be significantly higher than the New Delhi values. The morning as well as evening cortisol concentrations during the Austral winter were higher than the March values suggesting that cortisol rhythmicity was well maintained in Antarctica, albeit at a higher level. These observations indicated that the subtle changes in thyroid hormones during a prolonged stay at polar latitudes are related not only to the extreme cold but also to other factors such as physical activity, polar days and polar nights.
Adaptation, Physiological↗
Free and total thyroid hormones in humans at extreme altitude.
Alterations in circulatory levels of total T4 (TT4), total T3 (TT3), free T4 (FT4), free T3 (FT3), thyrotropin (TSH) and T3 uptake (T3U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized low-landers, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT3 and FT4 occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T3U was found to be higher at high altitudes.(ABSTRACT TRUNCATED AT 250 WORDS)
Acclimatization↗
Semiclassical and quantum mechanical analysis of the excitation function for the 130Te(p,n)130I reaction.
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Journal Article↗
Status of membrane lipids and amino acid transport in morphological mutants of Candida albicans.
The phospholipid composition of various morphological mutants of Candida albicans revealed a complete absence of phosphatidylinositol (PI) from plasma membranes of those cells which completely lacked mycelial growth. No other phospholipid was found to be specific to morphogenesis. The plasma membrane fractions isolated from mutants were more rigid than its wild type as was evident from their unsaturation index and fluorescence polarization measurements. The enhanced membrane rigidity of mutant cells was noted regardless whether the cells could grow only as mycelia or in their budding forms. Although some amino acids are considered to affect the morphological transition of C. albicans, this was not reflected in the transport activities of L-proline, L-alanine, L-lysine and L-glutamic acid.
Amino Acids↗
High membrane fluidity is related to NaCl stress in Candida membranefaciens.
The effect of hypersaline stress on the lipid composition of the salt-tolerant yeast Candida membranefaciens was studied. Fatty acid analyses of the plasma membrane showed a growth phase- and dose-dependent increase in the level of linolenic acid (C18:3) in 1.35 M NaCl-stressed cells. Palmitoleic acid (C16:1) was completely undetectable at all phases of the life cycle. Changes in the levels of other fatty acids were insignificant. The degree of unsaturation of fatty acids in the plasma membranes was higher in presence of 1.35 M NaCl. The fluorescence polarisation value of DPH (1,6-diphenyl- 1,3,5-hexatriene) in the spheroplasts of the stressed cells was lower as compared to the control cells, indicating the fact that a higher membrane fluidity favours osmotic adaptation against NaCl stress. Among different phospholipids, levels of Phosphatidylinositol and Phosphatidylethanolamine were elevated in the salt-adapted cells as compared to their controls. The levels of Phosphatidylcholine and cardiolipin did not change significantly in response to hypersaline stress. The study points out that hypersalinity signals affect the lipid composition which in turn affects the membrane fluidity of C. membranefaciens.
Candida↗
Multiple drug resistance in Candida albicans.
By functional complementation of a PDR 5 (pleiotropic drug resistance) null mutant of S. cerevisiae, we have recently cloned and sequenced a multidrug resistance gene CDR 1 (Candida Drug Resistance). Transformation by CDR 1 of a PDR 5 disrupted host hypersensitive to cycloheximide and chloramphenicol resulted in resistance to these as well as other unrelated drugs. The nucleotide sequence of CDR 1 revealed that, like PDR 5, it encodes a putative membrane pump belonging to the ABC superfamily. CDR 1 encodes a protein of 169.9 kDa whose predicted structural organisation is characterised by two homologous halves, each comprising a hydrophobic region, with a set of six transmembrane stretches, preceded by a hydrophilic binding fold. We now have evidence to suggest that there are several PDR homologues present in C. albicans which display multidrug resistance and a collateral sensitivity pattern different from PDR 5 and CDR 1. The functions of such genes and their products in the overall physiology of C. albicans is not yet established.
Antifungal Agents↗
Assignments of 1H, 15N, and 13C resonances for the backbone and side chains of the N-terminal domain of DNA polymerase beta. Determination of the secondary structure and tertiary contacts.
DNA polymerase beta consists of an N-terminal single-stranded DNA binding domain and a C-terminal catalytic domain separable by mild proteolysis [Kumar et al. (1990) J. Biol. Chem. 265, 2124-2131]. The N-terminal domain participates in template and gapped DNA recognition and contributes significantly to catalysis. The secondary structure and tertiary contacts within the cloned N-terminal domain (residues 2-87) of mammalian DNA polymerase beta have been determined using multidimensional NMR. Assignments of backbone 1H, 15N, and 13C resonances and side chain 1H and 13C resonances have been obtained from double- and triple-resonance 3D NMR experiments. The 13C-edited TOCSY experiment has allowed nearly complete assignments of 1H and 13C resonances within side chains. The 13C-edited NOESY experiment has been used for determination of medium- and long-range NOEs and a determination of tertiary contacts. The N-terminal domain is found to consist of four helices, helix-1 (15-26), helix-2 (36-47), helix-3 (56-61), and helix-4 (69-78), which on the basis of long-range NOEs are tightly packed of form a hydrophobic core. The remainder of the domain consists of two turns (48-51 and 62-65), an omega-type loop (27-35), and extended structure. The aromatic side chains of Y36, Y39, Y49, and F76 display tertiary contacts indicative of at least partial hydrophobic packing. The S30 and H34 residues which cross-link to single-stranded DNA [Prasad et al. (1993) J. Biol. Chem. 268, 15906-15911] are contained within the K27-K35 loop.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia.
Chromosome region 11q23 is involved in reciprocal chromosome translocations associated with human acute leukemias. These aberrations fuse the ALL-1 gene located at 11q23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. Here we report the cloning and characterization of the ALL-1 partner at 17q21, the AF17 gene. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus. The latter can be arranged in three zinc fingers and shows homology to a domain within the protein Br140 (peregrin). AF17 contains stretches of amino acids previously associated with domains involved in transcriptional repression or activation. Based on features of AF17 and of the proteins encoded by the other partner genes analyzed and in conjunction with other recent studies, we propose a model in which ALL-1 rearrangements result in loss of function of the gene. In this model, the partner polypeptide plays an accessory role either by repressing activity of the truncated ALL-1 protein or by blocking the function of the normal protein presumably present in the leukemic cells.
Amino Acid Sequence↗
Studies of gapped DNA substrate binding by mammalian DNA polymerase beta. Dependence on 5'-phosphate group.
Purified mammalian DNA polymerase beta (beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
Base Sequence↗
Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia.
DNA rearrangements caused by chromosome translocations between band 11q23 and various chromosomes can be detected by a single probe, B859, an 859-base pair complementary DNA fragment derived from the human ALL-1 gene. To try to understand why band 11q23 becomes a frequent target of the translocations, we have sequenced the entire breakpoint cluster region, a 8342-base pair BamHI genomic fragment delineated by B859. We found eight Alu repeats located within this region in the same orientation as the ALL-1 gene. We have also analyzed the sequences of the breakpoints in 10 patients with 6 different types of 11q23 aberration. In five patients the breaks coincided with Alu sequences on chromosome 11, but not on the partner chromosomes. Also, seven of the breaks occurred in the region delineated by exons 6 and 7, which is composed mainly of Alu sequences. In three patients topoisomerase II recognition site-like sequences, at different stringency levels, were identified at the breakpoints on chromosome 11. We conclude that while there is no specific sequence element present at all the breakpoints, the high density of Alu sequences in the breakpoint cluster region possibly makes the latter more prone to recombination events.
Acute Disease↗
Synthesis and structure-activity studies of N,N'-diarylguanidine derivatives. N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine: a new, selective noncompetitive NMDA receptor antagonist.
Diarylguanidines, acting as NMDA receptor ion channel site ligands, represent a new class of potential neuroprotective drugs. Several diarylguanidines structurally related to N,N'-di-o-tolylguanidine (DTG), a known selective sigma receptor ligand, were synthesized and evaluated in in vitro radioligand displacement assays, with rat or guinea pig brain membrane homogenates, using the NMDA receptor ion channel site specific radioligand [3H]-(+)-5(S)-methyl-10(R),11-dihydro-5H-dibenzo[a,d]cyclohepten-5 ,10- imine (MK-801, 3), and the sigma receptor-specific radioligand [3H]-di-o-tolylguanidine (DTG, 5). This paper presents the structure-activity relationships leading to novel tri- and tetrasubstituted guanidines, which exhibit high selectivity for NMDA receptor ion channel sites and weak or negligible affinity for sigma receptors. The in vitro binding results from symmetrically substituted diphenylguanidines indicated that compounds having ortho or meta substituents (with respect to the position of the guanidine nitrogen) on the phenyl rings showed greater affinity for the NMDA receptor ion channel site compared with para-substituted derivatives. Among the group of ring substituents studied for symmetrical diarylguanidines, an isopropyl group was preferred at the ortho position and an ethyl group was preferred at the meta position. Several unsymmetrical guanidines containing a naphthalene ring on one nitrogen atom and an ortho- or a meta-substituted phenyl ring on the second nitrogen atom, e.g., N-1-naphthyl-N'-(3-ethylphenyl)guanidine (36), showed a 3-5-fold increase in affinity for the NMDA receptor ion channel site and no change in sigma receptor affinity compared to the respective symmetrical counterparts. Additional small substituents on the guanidine nitrogen atoms bearing the aryl rings resulted in tri- and tetrasubstituted guanidine derivatives which retained affinity for NMDA receptor ion channel sites but exhibited a significant reduction in their affinities for sigma receptors. For example, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) showed high affinity for the NMDA receptor ion channel site (IC50 = 36 nM vs [3H]-3) and low affinity for sigma receptors (IC50 = 2540 nM vs [3H]-5). Selectivity for the NMDA receptor ion channel sites over sigma receptors appears to be dependent upon the structure of the additional substituents on the guanidine nitrogen atoms bearing the aryl groups. Methyl and ethyl substituents are most preferred in the tri- and tetrasubstituted diarylguanidines. The trisubstituted guanidine, N-1-naphthyl-N'-(3-ethylphenyl)-N'-methylguanidine (40) and its close analogues showed good in vivo neuroprotection and are potential neuroprotective drug candidates for the treatment of stroke and other neurodegenerative disorders.
Animals↗
Molecular characterization of an enterotoxin from Salmonella typhimurium.
In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb ClaI/PstI DNA fragment, was cloned from a genomic library of Salmonella. Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29,073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli. The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.
Amino Acid Sequence↗