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Biomedical subjects

P Roy

Publications and source records attributed to P Roy.

At least 181 records · Page 10Linked to original sources
PubMed1995

Synthesis of bluetongue virus chimeric VP3 molecules and their interactions with VP7 protein to assemble into virus core-like particles.

Bluetongue virus (BTV) core-like particles (CLPs) are formed in the cytoplasm of insect cells when only two major proteins (VP3 and VP7) of the BTV core are expressed by baculovirus vectors (T. J. French and P. Roy, 1990, J. Virol. 64, 1530-1536). We have recently reported that five small internal deletion mutants of VP3 form CLPs when provided with unmodified VP7 protein (D1-5; S. Tanaka and P. Roy, 1994, J. Virol. 68, 2795-2802). To investigate whether foreign sequences can be inserted into VP3 and to determine their effect on CLP formation, three of these internal regions (D1, D2, and D5), as well as the carboxy terminus, were modified to create unique restriction enzyme sites, thereby replacing VP3 coding regions with shorter synthetic sequences. Each modified VP3 gene was used to generate baculovirus expression vectors (D1I, D2I, D5I, and VP3C, respectively). Other than the D5I mutant, the mutants formed CLPs when expressed in the presence of VP7. Subsequently, T7 tag epitopes were inserted into the D1I, D2I, and VP3C restriction sites and recombinant baculoviruses were generated to express chimeric VP3 proteins (VP3D1IT, VP3D2IT, and VP3CT). Each chimeric protein retained the ability to form CLPs when VP7 protein was provided. In another construction an immunogenic sequence representing a bovine leukemia virus (BLV) glycoprotein peptide was incorporated into the carboxy terminus of VP3 and the derived CLPs were used to raise antibodies that reacted with the BLV antigen. The results suggest that the VP3 molecule can accommodate foreign sequences at certain sites without perturbing their ability to form CLPs with VP7.

Amino Acid Sequence↗
PubMed1995

Crystal structure of SIV matrix antigen and implications for virus assembly.

Simian immunodeficiency virus (SIV) is closely related to human immunodeficiency virus (HIV), their matrix antigens (MAs) sharing some 50% sequence identity. MA is a component of Pr55Gag, the sole protein required for assembly of the virion shell. MA targets Pr55 to the plasma membrane, and facilitates incorporation of the virus envelope protein and assembly of the Pr55Gag shell. Cleavage of Pr55 by the viral protease produces the mature protein of relative molecular mass 17-18K, which underlies the host-derived membrane and is important in both virus entry and nuclear localization of the virion core. Here we report the crystal structure of SIV MA. The molecule forms a trimer consistent with oligomerization in vitro, the observed virion architecture, and various biological properties of MA.

Amino Acid Sequence↗
PubMed1995

5-Nitrofuran derivatives of fatty acid hydrazides induce differentiation in human myeloid leukaemic cell lines.

Compounds formed by 5-nitrofuran with hydrazides of formic, acetic and propionic acids, hereafter respectively known as SBF, SBA and SBP have been used to evaluate the differentiation-inducing properties on two established myeloid leukaemic cell lines ML-2 and EOL-1. SBP is found to be the most effective as an antineoplastic agent amongst the three. Induction of differentiation observed are in the order SBP > SBA > SBF, as assessed by morphology, NBT-reducing activity and surface marker antigens of the treated cells. Induction of differentiation of ML-2 and EOL-1 cells by the most effective compound, SBP (3 microM), is accompanied by perturbation of the cell cycle, with most of the cells accumulating in the G0-G1, phase. Inhibition of DNA synthesis occurs while protein and RNA synthesis remain practically unchanged.

Acetates↗
PubMed1995

High-level expression of five foreign genes by a single recombinant baculovirus.

Co-infection with several viruses is often used to achieve simultaneous expression of several proteins. From co-infections involving several viruses, the ratio of proteins synthesized in individual cells can be very variable. This is disadvantageous where proteins are needed to interact to provide a maximum yield of a complex product. Multiple-gene-expression vectors offer an alternative to co-infections. They enable reproducible ratios of products to be provided in each infected cell. Until recently, multigene-expression vectors have only been developed to make two proteins simultaneously. Here, we describe the generation of a single recombinant baculovirus synthesising up to five foreign proteins with a fixed ratio comparable to the ratio during the synthesis of native proteins.

Animals↗
PubMed1995

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.

Journal Article↗
PubMed1995

The crystal structure of bluetongue virus VP7.

Bluetongue virus (BTV), a representative of the orbivirus genus of the Reoviridae, is considerably larger (at 80 nm across), and structurally more complex, than any virus for which we have comprehensive structural information. Orbiviruses infect mammalian hosts through insect vectors and cause economically important diseases of domesticated animals. They possess a segmented double-stranded RNA genome within a capsid composed of four major types of polypeptide chains. An outer layer of VP2 and VP5 is removed as the virus enters the target cell, to leave an intact core within the cell. This core is 70 nm across and composed of 780 copies of VP7 (M(r) 38K) that, as trimers, form 260 'bristly' capsomeres clothing an inner scaffold constructed from VP3 (M(r) 103K). We report here the crystal structure of VP7 from BTV serotype 10, which reveals a molecular architecture not seen previously in viral structural proteins. Each subunit consists of two domains, one a beta-sandwich, the other a bundle of alpha-helices, and a short carboxy-terminal arm which might tie trimers together during capsid formation. A concentration of methionine residues at the core of the molecule could provide plasticity, relieving structural mismatches during assembly.

Amino Acid Sequence↗
PubMed1995

Hyperaggregation of platelets in intestinal tuberculosis: role of platelets in chronic inflammation.

Eighty-eight percent (38/43) patients of intestinal tuberculosis showed significant hyperaggregation of platelets (P < 0.001). Serum and plasma from 15 patients when incubated with normal platelets caused hyperaggregation. Gel filtered platelets from 2 patients suspended in normal plasma showed hyperaggregation of platelets with arachidonic acid. Tubercular protein had no effect on platelet aggregation. A role of hyperactive platelets in chronic inflammatory response is discussed.

Adolescent↗
PubMed1995

Lipoxygenase-mediated glutathione oxidation and superoxide generation.

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a Km of 1.49 mM for GSH and Vmax of 1.35 +/- 0.02 mumoles/min/nmole of enzyme. Besides LA, arachidonic and gamma-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3'-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 +/- 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.

Fatty Acids, Nonesterified↗
PubMed1995

The use of African horse sickness virus NS3 protein, expressed in bacteria, as a marker to differentiate infected from vaccinated horses.

Segment 10 of the double-stranded RNA (dsRNA) genome from African horse sickness virus serotype 4 (AHSV-4) was cloned and sequenced. The sequence of the coding region showed a total length of 667 bp. Nucleotide comparisons showed a 95% sequence similarity between serotypes 4 and 9, and 76% between serotypes 4 and 3. cDNA clones containing the coding region were cloned in the vector pET3xb and expressed in Escherichia coli. The NS3 gene product was synthesised at very high level as an insoluble fusion protein. The recombinant protein was used in a differential ELISA to distinguish horses that were infected with AHSV-4 or vaccinated with live-modified virus from those vaccinated with a purified inactivated vaccine. The results obtained indicate that recombinant NS3 can indeed differentiate between infected and vaccinated animals implying that this recombinant could be developed as a diagnostic reagent, and it would allow the mobility of vaccinated horses. Thus, economical losses associated with this disease could be avoided.

African Horse Sickness↗
PubMed1995

Is neuroblastoma screening evaluation needed and feasible?

Despite the five million children who have been screened for neuroblastoma in Japan through detection of catecholamine metabolites, it is still uncertain whether screening for this disease is beneficial. The Japanese study has clearly indicated that screening at 6 months or earlier leads to heavy overdiagnosis. It is shown in this paper that screening at a later age may give the same reduction in mortality with possibly less overdiagnosis. However, it is estimated that, even with two screens at 12 and 18 months, the reduction in mortality would not greatly exceed 25%, under realistic hypotheses on the length of the preclinical phase of the disease. The evaluation of the efficacy of this screening strategy would need the recruitment of half a million children per year over 5-7 years and the follow-up of an equal number of controls. Such a trial would improve our knowledge of the natural history of the disease and might help to answer some questions raised recently regarding its biological heterogeneity.

Age Factors↗
PubMed1995

Characterization and modification of the carboxy-terminal sequences of bluetongue virus type 10 NS1 protein in relation to tubule formation and location of an antigenic epitope in the vicinity of the carboxy terminus of the protein.

Bluetongue virus produces large numbers of tubules during infection. The tubules are formed from a 552-amino-acid, 64-kDa NS1 protein encoded by the viral double-stranded RNA segment M6. A series of deletion and extension mutants of bluetongue virus serotype 10 NS1 has been generated and expressed in insect cells in order to identify the carboxy-terminal components of the protein which are important for tubule formation. The mutants AcCT5 and AcCT10, lacking 5 and 10 of the carboxy-terminal residues, respectively, were prepared. By analyzing their abilities to form tubules, it was shown that AcCT5 was capable of this function whereas AcCT10 was not, indicating that the last five amino acids are not strongly involved in NS1 tubule formation. Extension mutants including foreign antigenic sequences involving up to 16 amino acids added to the C terminus of NS1 were shown to form tubules, although an extension of 19 amino acids inhibited tubule formation. Analysis of a panel of monoclonal antibodies has established that an NS1 antigenic site is located near the carboxy terminus of the protein. It appears to be exposed on the surface of tubules. The opportunities to develop new vaccines using recombinant NS1 to deliver foreign epitopes are discussed.

Amino Acid Sequence↗
PubMed1995

BCL6 gene rearrangement and other cytogenetic abnormalities in diffuse large cell lymphoma.

Evidence for rearrangement of the BCL6 gene at 3q27 has been documented in 20-30% diffuse lymphomas with a large cell component (DLLC), and was found to be of prognostic significance at the time of diagnosis. To incorporate these observations into current cytogenetic and clinical prognostic models, 76 cases of DLLC with known BCL6 status were analyzed. Cytogenetic indicators of progression, including trisomy 7, trisomy 12, del(6)(q21q25), and structural alterations of 17p were less frequent in BCL6 rearranged DLLC compared to BCL6 germline tumors. Despite a 93% overall survival at median follow-up of 30 months, a trend for continued relapse resulted in a projected freedom from progression for the BCL6 rearranged cohort of 66% at 4 years, compared to 39% for the BCL6 germline cohort. Six cases among the BCL6 rearranged group lacked additional cytogenetic indicators of progression and remained free of disease at follow-up in excess of 7 years, whereas BCL6 rearranged cases with increasing numbers of cytogenetic aberrations showed decreased intervals free from progression of disease. These results suggest that BCL6 rearrangement should be combined with other known clinical and cytogenetic indicators in prognostic analyses of patients with DLLC.

Chromosome Aberrations↗
PubMed1994

Apo E allele frequencies in Alzheimer's disease, Lewy body dementia, Alzheimer's disease with cerebrovascular disease and vascular dementia.

Apolipoprotein (Apo E) was genotyped using a fluorescent PCR-RFLP assay in 187 patients with a probable or possible clinical diagnosis of sporadic Alzheimer's disease (AD) and in 166 autopsied patients with dementia (21 presenile AD, 70 senile AD, 18 Lewy body dementia (LBD), 38 AD with cerebrovascular disease (AD-CVD), 19 vascular dementia). The relative epsilon 4 allele frequency was 0.472 in LBD, 0.513 in AD-CVD, 0.405 in presenile AD, 0.364 in senile AD, and 0.079 in vascular dementia. The relative epsilon 2 allele frequency was 0.211 in vascular dementia, 0.083 in LBD, 0.047 in presenile AD, 0.100 in senile AD and 0.039 in AD with CVD. We infer that apo E is a major risk factor for structural phenotypes of dementia involving AD, alone or in conjunction with another pathology. In addition, the epsilon 2 allele is likely to represent a risk factor for vascular morbidity, as the relative epsilon 2 allele frequency was 0.211 in patients with vascular dementia compared with 0.144 in elderly controls.

Adolescent↗