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Biomedical subjects

V Prasad

Publications and source records attributed to V Prasad.

At least 73 records · Page 4Linked to original sources
PubMed1999

Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex.

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

DNA, Viral↗
PubMed1998

Podophyllotoxin and nocodazole counter the effect of IKP104 on tubulin decay.

Tubulin, the subunit protein of microtubules, undergoes a time-dependent loss of functional properties known as decay. We have previously shown that the drug 2-(4-fluorophenyl)- -(2-chloro-3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP104) accelerates decay, but that in the presence of colchicine, IKP104 becomes a stabilizer of tubulin. To see if this is due to conformational effects specific to colchicine or simply to occupancy at the colchicine site, we examined the effects of nocodazole and podophyllotoxin, two well-known competitive inhibitors of colchicine for binding to tubulin, on IKP104's acceleration of decay. We found that podophyllotoxin abolished IKP104's accelerating effect and, like colchicine, turned it into a stabilizer of tubulin. Nocodazole's effects were similar to those of podophyllotoxin and colchicine, in that it abolished IKP104-induced enhancement of decay; however, in the presence of nocodazole, IKP104 caused little or no stabilization of tubulin. Since colchicine, nocodazole, and podophyllotoxin have very different interactions with tubulin, but all inhibit the IKP104-induced enhancement of decay, our findings suggest that this inhibition arises from occupancy of the colchicine site rather than from a direct conformational effect of these two drugs.

Alkylation↗
PubMed1997

Effect of yogurt and bifidus yogurt fortified with skim milk powder, condensed whey and lactose-hydrolysed condensed whey on serum cholesterol and triacylglycerol levels in rats.

The possible hypocholesterolaemic properties of milk and fermented milk products have been investigated in groups of albino rats given a basal diet, basal diet plus cholesterol, and basal diet plus cholesterol together with whole milk or standard or bifidus yogurt. The yogurts were fortified with skim milk powder, condensed whey or lactose-hydrolysed condensed whey. After 30 d, triacylglycerols, total cholesterol, HDL-cholesterol and LDL-cholesterol were measured in serum. Whole milk and ordinary yogurt had no hypocholesterolaemic effect, but standard yogurt containing lactose-hydrolysed condensed whey and all bifidus yogurts lowered serum cholesterol. In general, yogurts changed HDL-cholesterol little, but tended to raise triacylglycerols. There was marked lowering of LDL-cholesterol in rats given either type of yogurt fortified with whey proteins. This study has demonstrated in a rat model that bifidus yogurts and yogurts fortified with whey proteins can reduce total and LDL-cholesterol, and suggests that if they have the same effect in human subjects they have potential value in cholesterol-lowering diets.

Animals↗
PubMed1997

Hypothalamic-pituitary-adrenal function following cranial irradiation.

We assessed the effect of cranial irradiation on hypothalamic-pituitary (HP)-adrenal function in 17 patients (12 females, 5 males) treated with cranial/ craniospinal irradiation for acute leukemia (2 patients) or tumors distant from the hypothalamus and pituitary (8 medulloblastoma, 3 astrocytoma, 3 rhabdomyosarcoma, 1 ependymoma). Estimated doses of radiation (RT) to the HP region ranged from 18 to 72 Gy. Thirteen of seventeen patients were also treated with chemotherapy. Patients were a median of 3.75 years of age (1.5-19 years) at diagnosis and were studied at a median of 5 years (0.1-20 years) after RT. Patients received corticotropin-releasing factor (oCRF, 1 microgram/kg i.v.), and sampling for cortisol and ACTH levels was performed at -15, 0, 15, 30, 60, 90 and 120 min. The-5- and 0-min levels were combined for a standardized baseline value (Base). Cortisol levels at 0, Base, 30 and 120 min, as well as the peak cortisol response, were significantly lower in the patients. Twelve of seventeen patients' peak cortisol levels fell below the normal range. The patients' mean integrated values for cortisol (area under the curve) were not, however, different from controls. The ACTH responses to oCRF did not differ between patients and controls. No relationship was observed between ACTH or cortisol responses and the time elapsed from treatment or dose of HP RT. Further, in 10 of 12 patients, 0-min dehydroepiandrosterone sulfate levels were lower than the expected normal mean levels for age, sex and pubertal status, and in 4 of these 10 patients the values were below the normal range. These data suggest that some patients treated with HP RT may be at risk for adrenal insufficiency.

Adolescent↗
PubMed1996

A systemic antiviral resistance-inducing protein isolated from Clerodendrum inerme Gaertn. is a polynucleotide:adenosine glycosidase (ribosome-inactivating protein).

Two systemic antiviral resistance-inducing proteins, CIP-29 and CIP-34, isolated from Clerodendrum inerme Gaertn. leaves, were tested for ribosome-inactivating properties. It was found that CIP-29 has the characteristics of a polynucleotide:adenosine glycosidase (ribosome-inactivating protein), in that it inhibits protein synthesis both in cell-free systems and, at higher concentrations, in cells, and releases adenine from ribosomes, RNA, poly(A) and DNA. As compared with other known RIPs, CIP-29 deadenylates DNA at a high rate, and induces systemic antiviral resistance in susceptible plants.

Adenine↗
PubMed1995

Interaction of bovine brain tubulin with the 4(1H)-pyrizinone derivative IKP104, an antimitotic drug with a complex set of effects on the conformational stability of the tubulin molecule.

The ligands of tubulin have proved to be excellent probes for the conformation of the tubulin molecule. The most varied in their effects on tubulin are those ligands which are competitive or noncompetitive inhibitors of vinblastine binding. The 4(H)-pyrizinone derivative 2-(4-fluorophenyl)-1-(2-chloro- 3,5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone [sequence: see text] (IKP104) is a novel antimitotic drug which inhibits microtubule assembly in vitro and in vivo and polymerizes tubulin into spiral filaments. Using a fluorescence assay, we found that IKP104 appears to bind to tubulin at two classes of site, differing in affinity. IKP104 also blocks formation of an intrachain cross-link in beta-tubulin, induced by N,N"-ethylenebis(iodoacetamide), linking Cys12 to either Cys201 or Cys211. IKP104 appears to belong to that group of tubulin ligands which includes vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, and halichondrin B. An unusual effect of IKP104 is that it greatly enhances the decay or apparent unfolding or opening of the tubulin molecule. The sulfhydryl titer of tubulin is doubled and the exposure of hydrophobic areas on the tubulin molecule is tripled by IKP104. These effects of IKP104 are counteracted by vinblastine, maytansine, and phomopsin A, suggesting that IKP104 may be competing with these other drugs for binding to tubulin. However, the effects are also counteracted by colchicine and podophyllotoxin, implying a more complex effect, namely, that IKP104 and colchicine, even when both are bound to tubulin, are competing for their effects on the same domain of tubulin. Surprisingly, when IKP104 is used in conjunction with colchicine, binding of colchicine to tubulin is strongly stabilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1994

Molecular characterization of a novel tobacco pathogenesis-related (PR) protein: a new plant chitinase/lysozyme.

A new PR (pathogenesis-related) protein was isolated from tobacco leaves (Nicotiana tabacum cv. Samsun NN), reacting hypersensitively to tobacco mosaic virus (TMV), by zinc chelate chromatography and was therefore named Pz. Its reactivity toward several lectins indicated the presence of bound sugar residues. From the amino acid sequence of tryptic peptides, Oligonucleotide primers were derived which allowed the synthesis of Pz cDNA by PCR. Using this cDNA as probe, near full-length clones were isolated from a library made from poly(A)+ RNA purified from TMV-infected leaves. Sequence analysis revealed similarities with chitinases/lysozymes of various origins and the purified protein was, indeed, shown to hydrolyse different N-acetylglucosamine-containing substrates. Comparison of peptide and cDNA sequences indicated that Pz protein is synthesized as a pre-pro-protein, a seven-amino acid C-terminal peptide probably being involved in the vacuolar targeting of the protein. Pz mRNA and protein were demonstrated to accumulate strongly in TMV-infected tobacco leaves. Pz transcripts were also found in various tissues of healthy plants, indicating that Pz gene expression is developmentally regulated.

Amino Acid Sequence↗
PubMed1994

Interaction of ustiloxin A with bovine brain tubulin.

Ustiloxin A is a modified peptide derived from false smut balls on rice panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin A is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomopsin A, we examined its interaction with tubulin and compared the results with those obtained with phomopsin A and dolastatin 10, both of which were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain cross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, phomopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylation of tubulin by iodo[14C]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent as inhibitor of alkylation as ustiloxin A, whereas maytansine, halichondrin B and homohalichondrin B have little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this respect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobic areas and to halichondrin B which enhances exposure. Lastly, ustiloxin A strongly stabilized the binding of [3H]colchicine to tubulin. The combination of ustiloxin A with cholchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomopsin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong stabilization of the tubulin structure may require a short segment of hydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifically the large ring of ustiloxin A and phomopsin A, may serve to place this sequence in an appropriate conformation to interact with tubulin.

Amino Acid Sequence↗
PubMed1994

Removal of the projection domain of microtubule-associated protein 2 alters its interaction with tubulin.

Microtubule-associated proteins (MAPs) can promote microtubule assembly in vitro. One of these MAPs (MAP2) consists of a short promoter domain which binds to the microtubule and promotes assembly and a long projection domain which projects out from the microtubule and may interact with other cytoskeletal elements. We have previously shown that MAP2 and another MAP, tau, differ in their interactions with tubulin in that tau, but not MAP2, promotes extensive aggregation of tubulin into spiral clusters in the presence of vinblastine and that microtuubles formed with MAP2 are more resistant than those formed with tau to the antimitotic drug maytansine [Luduena, R. F., et al. (1984), J. Biol. Chem. 259, 12890-12898; Fellous, A., et al. (1985), Cancer Res. 45, 5004-5010]. Here we have used chymotryptic digestion to remove the projection domain of MAP2 and examined the interaction of the digested MAP2 (ctMAP2) with tubulin in the presence of vinblastine and maytansine. We have found that ctMAP2 behaves very much like tau, but not like undigested MAP2, in the presence of vinblastine, in that ctMAP2 causes tubulin to polymerize into large clusters of spirals. In contrast, microtubule assembly in the presence of ctMAP2 is much more resistant to maytansine inhibition than is assembly in the presence of tau or undigested MAP2. Our results suggest that the projection domain of MAP2 may play a role in the interaction of tubulin with MAP2 during microtubule assembly.

Binding Sites↗
PubMed1993

Interaction of halichondrin B and homohalichondrin B with bovine brain tubulin.

Halichondrin B is a polyether macrolide of marine origin which binds to tubulin and inhibits microtubule assembly in vitro and in vivo. As is the case with phomopsin A and dolastatin 10, halichondrin B is a non-competitive inhibitor of vinblastine binding to tubulin. Analogous to maytansine, which by contrast is a competitive inhibitor of vinblastine binding, halichondrin B has no effect on colchicine binding, which is greatly stabilized by phomopsin A and dolastatin 10, but not by maytansine. We have previously developed assays which allow sensitive discrimination among the interactions of various ligands with tubulin, and examined the effects of ligands on the reactivity of tubulin sulfhydryl groups and the exposure of hydrophobic areas on the surface of the tubulin molecule. To classify the nature of the interaction between halichondrin B and tubulin, in this study we examined the effects of halichondrin B and its closely related analogue, homohalichondrin B, by these assays. We found that: (1) halichondrin B and homohalichondrin B both inhibited formation of an intra-chain cross-link between two sulfhydryl groups in beta-tubulin, as do phomopsin A, dolastatin 10, maytansine, and vinblastine; (2) halichondrin B resembles maytansine in that it had no effect on alkylation of tubulin sulfhydryl groups by iodoacetamide, unlike phomopsin A, dolastatin 10 and vinblastine, all of which inhibit alkylation; (3) halichondrin B differs from other anti-mitotic drugs in that it enhanced exposure of hydrophobic areas on tubulin; (4) homohalichondrin B, like maytansine and in contrast to phomopsin A, dolastatin 10 and vinblastine, had no effect on exposure of hydrophobic areas; and (5) homohalichondrin B, contrary to maytansine, inhibited alkylation of tubulin sulfhydryl groups in the presence of GTP and MgCl2. In their interactions with the tubulin molecule, halichondrin B and homohalichondrin B appear to have unique conformational effects which differ from those of other drugs and also from the effects of each other as well.

Alkylation↗
PubMed1993

Plant 'pathogenesis-related' proteins and their role in defense against pathogens.

The hypersensitive reaction to a pathogen is one of the most efficient defense mechanisms in nature and leads to the induction of numerous plant genes encoding defense proteins. These proteins include: 1) structural proteins that are incorporated into the extracellular matrix and participate in the confinement of the pathogen; 2) enzymes of secondary metabolism, for instance those of the biosynthesis of plant antibiotics; 3) pathogenesis-related (PR) proteins which represent major quantitative changes in soluble protein during the defense response. The PRs have typical physicochemical properties that enable them to resist to acidic pH and proteolytic cleavage and thus survive in the harsh environments where they occur: vacuolar compartment or cell wall or intercellular spaces. Since the discovery of the first PRs in tobacco many other similar proteins have been isolated from tobacco but also from other plant species, including dicots and monocots, the widest range being characterized from hypersensitively reacting tobacco. Based first on serological properties and later on sequence data, the tobacco PRs have been classified in five major groups. Group PR-1 contains the first discovered PRs of 15-17 kDa molecular mass, whose biological activity is still unknown, but some members have been shown recently to have antifungal activity. Group PR-2 contains three structurally distinct classes of 1,3-beta-glucanases, with acidic and basic counterparts, with dramatically different specific activity towards linear 1,3-beta-glucans and with different substrate specificity. Group PR-3 consists of various chitinases-lysozymes that belong to three distinct classes, are vacuolar or extracellular, and exhibit differential chitinase and lysozyme activities. Some of them, either alone or in combination with 1,3-beta-glucanases, have been shown to be antifungal in vitro and in vivo (transgenic plants), probably by hydrolysing their substrates as structural components in the fungal cell wall. Group PR-4 is the less studied, and in tobacco contains four members of 13-14.5 kDa of unknown activity and function. Group PR-5 contains acidic-neutral and very basic members with extracellular and vacuolar localization, respectively, and all members show sequence similarity to the sweet-tasting protein thaumatin. Several members of the PR-5 group from tobacco and other plant species were shown to display significant in vitro activity of inhibiting hyphal growth or spore germination of various fungi probably by a membrane permeabilizing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

Chitinases↗
PubMed1992

Interaction of dolastatin 10 with bovine brain tubulin.

Dolastatin 10 is an unusual peptide of marine origin which binds to tubulin in the vinblastine/maytansine/phomopsin-binding region and potently inhibits mitosis. Using N,N'-ethylenebis(iodoacetamide) (EBI) and iodo[14C]acetamide as probes for the effects of ligands on the thiol groups of tubulin, we found that dolastatin 10 has effects on the sulfhydryls indistinguishable from those of phomopsin A but quite different from those of vinblastine and maytansine. Using the binding of bis-5,5'-[8-(N-phenyl)aminonaphthalene-1-sulfonic acid] (BisANS) as a measure of tubulin decay, we found that dolastatin 10 resembled phomopsin A in that decay was not detectable by this assay in its presence. Interestingly, both otherwise very different peptides are among the most effective inhibitors of tubulin decay yet discovered.

Alkylation↗
PubMed1992

Temperature sensitivity of vinblastine-induced tubulin polymerization in the presence of microtubule-associated proteins.

The antitumor drug vinblastine has been a useful probe for examining the interaction of tubulin with the microtubule-associated proteins (MAPs), specifically with tau and MAP 2. Although tau and MAP 2 can stimulate microtubule assembly in vitro, their specific interactions with tubulin are known to differ. For example, in the presence of vinblastine, both tau and MAP 2 cause tubulin to form spirals, but tau causes formation of clustered spirals of high turbidity, while MAP 2 causes formation of loose spirals of low turbidity [Ludueña et al., J. Biol. Chem. 259, 12890-12898 (1984)]. Although cold temperatures can inhibit microtubule assembly, cold has no effect on vinblastine-induced tubulin spiral formation. Consequently, we used the vinblastine-tubulin system to examine the interactions of tau and MAP 2 with tubulin at low temperatures. We found that tau-tubulin-vinblastine complexes form about as well at 0 degree C as at 37 degrees C. In contrast, MAP 2-tubulin-vinblastine complexes form much less well at 0 degrees C than at 37 degrees C. We find, however, that MAP 2, at 0 degrees C, will strongly inhibit, and even reverse, formation of the tau-tubulin-vinblastine complex. This suggests that the temperature-sensitive factor is the MAP 2-stimulated tubulin-tubulin interaction rather than the MAP 2-tubulin interaction per se; this raises the possibility that the tubulin-tubulin interactions stimulated by tau differ in their temperature sensitivity from those stimulated by MAP 2.

Animals↗
PubMed1992

Hemispheric priming: effect of response mode on a position replication task.

Ten male and ten female dextral subjects performed a kinesthetic position replication task under two response mode conditions; in one block, subjects indicated their decision by a lateralized verbal response and in the other block, by a midline chin-lift response. Response mode was found to interact with arm, excursion length, end position, and sex. This interaction is interpreted to reflect the influence of verbal preparation on the relative balance between a left hemisphere-biased strategy of kinesthetic positioning and a right hemisphere-biased strategy involving a spatial reference system.

Acoustic Stimulation↗
PubMed1991

Further experiences of Küntscher's nailing for tibial shaft fractures.

We present the experience of managing 429 fractures of the tibia fixed with straight Küntscher rods. The overall impression is quite encouraging because this method involves a short and easy operation with limited exposure and provides sound fixation. Complications have been negligible.

Adolescent↗