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Biomedical subjects

P Roy

Publications and source records attributed to P Roy.

At least 199 records · Page 11Linked to original sources
PubMed1994

Superoxide generation by lipoxygenase in the presence of NADH and NADPH.

The ability of soybean lipoxygenase to mediate NAD(P)H oxidation and concomitant superoxide generation in the presence of linoleic acid was examined. At optimum pH of 8.3, lipoxygenase oxidized both NADH and NADPH in the presence of 700 microM linoleic acid. The oxidation of NAD(P)H was biphasic. The initial rates of NADH and NADPH oxidation were 130 and 140 nmoles/min/nmole of enzyme respectively and the corresponding final rates were 344 and 350 nmoles/min/nmole of enzyme. The apparent Km values calculated for NADH and NADPH oxidation were 13 microM and 117 microM respectively. NAD(P)H oxidation was accompanied by the reduction of either ferricytochrome c or nitroblue tetrazolium (NBT) which can be abolished (approx. 85%) by superoxide dismutase (SOD) suggesting the generation of superoxide anion radicals. Under optimal conditions, the rates of superoxide generation, measured as the SOD-inhibitable reduction of ferricytochrome c, were 325 and 235 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Under identical experimental conditions, the SOD-inhibitable NBT reduction rates were 308 and 210 nmoles/min/nmole of enzyme for NADH and NADPH respectively. Both NADH and NADPH could be regenerated after oxidation using the appropriate dehydrogenases. These results strongly suggest that lipoxygenase not only generates lipid hydroperoxides but can also generate superoxide via oxidation of pyridine nucleotides and may, therefore, significantly contribute to oxidative stress in cells.

Alcohol Dehydrogenase↗
PubMed1994

Crystallization and preliminary X-ray investigation of recombinant simian immunodeficiency virus matrix protein.

Simian immunodeficiency virus matrix protein has been crystallized from Tris-HCl buffer with polyethylene glycol and isopropanol as precipitants. The crystals belong to space group C2 with unit cell dimensions a = 69.9 A, b = 115.2 A, c = 32.5 A, beta = 108.1 degrees and diffract X rays to a minimum Bragg spacing of 2 A. These crystals appear suitable for a high resolution structure analysis.

Crystallization↗
PubMed1994

Risk factors for gastrointestinal bleeding in critically ill patients. Canadian Critical Care Trials Group.

BACKGROUND: The efficacy of prophylaxis against stress ulcers in preventing gastrointestinal bleeding in critically ill patients has led to its widespread use. The side effects and cost of prophylaxis, however, necessitate targeting preventive therapy to those patients most likely to benefit. METHODS: We conducted a prospective multicenter cohort study in which we evaluated potential risk factors for stress ulceration in patients admitted to intensive care units and documented the occurrence of clinically important gastrointestinal bleeding (defined as overt bleeding in association with hemodynamic compromise or the need for blood transfusion). RESULTS: Of 2252 patients, 33 (1.5 percent; 95 percent confidence interval, 1.0 to 2.1 percent) had clinically important bleeding. Two strong independent risk factors for bleeding were identified: respiratory failure (odds ratio, 15.6) and coagulopathy (odds ratio, 4.3). Of 847 patients who had one or both of these risk factors, 31 (3.7 percent; 95 percent confidence interval, 2.5 to 5.2 percent) had clinically important bleeding. Of 1405 patients without these risk factors, 2 (0.1 percent; 95 percent confidence interval, 0.02 to 0.5 percent) had clinically important bleeding. The mortality rate was 48.5 percent in the group with bleeding and 9.1 percent in the group without bleeding (P < 0.001). CONCLUSIONS: Few critically ill patients have clinically important gastrointestinal bleeding, and therefore prophylaxis against stress ulcers can be safely withheld from critically ill patients unless they have coagulopathy or require mechanical ventilation.

Aged↗
PubMed1994

Complementation of human immunodeficiency virus (HIV-1) gag particle formation.

The human immunodeficiency virus gag precursor protein Pr55Gag exhibits the ability of particle assembly when expressed using recombinant baculoviruses. In order to delineate the sequences required for particle formation, two mutants of Gag (D1 and D2) were constructed in which 10 amino acids within the CA domain were deleted. Both mutants yielded stable high levels of Gag antigen following expression in Spodoptera frugiperda insect cells. Electron microscopy of sections through infected cells revealed that neither mutant was able to assemble particles although targeting of the protein to the plasma membrane still occurred. The Gag antigen that accumulated beneath the plasma membrane exhibited distinctive morphologies when compared to each other and to parental (Pr46Gag) particles. Particle assembly was rescued when S. frugiperda cells were coinfected with both AcD1 and AcD2 viruses, or with AcD1 and a carboxyl-terminal deletion of Gag (Pr41.5) which was previously shown not to form particles (J.B.M., D.J. Hockley, M.V. Nermot, and I.M. Jones, 1992, J. Gen. Virol. 73, 3079-3086). The genetic complementation of Gag-driven assembly is discussed.

Amino Acid Sequence↗
PubMed1994

Expression and characterization of the two outer capsid proteins of African horsesickness virus: the role of VP2 in virus neutralization.

African horsesickness virus (AHSV) is a gnat-transmitted member of the Orbivirus genus of the Reoviridae family. The virus has a genome of 10 double-stranded RNA species (L1-L3, M4-M6, S7-S10). The L2 and M6 genes of AHSV serotype 4 (AHSV-4) which encode the outer capsid proteins VP2 and VP5, respectively, were inserted into recombinant baculoviruses downstream of the baculovirus polyhedrin, or p10 promoters. Recombinant baculoviruses expressing VP2, VP5, or VP2 and VP5 proteins of AHSV-4 were isolated. The expressed AHSV proteins were similar in size and antigenic properties to those of viral AHSV-4. Expressed VP2 and VP5 proteins were purified to homogeneity and utilized to differentiate sera from vaccinated and infected horses. Antigens were also used to determine whether any other AHSV serotypes are related to AHSV-4. The results indicated that AHSV-4 is distantly related to some serotypes (e.g., AHSV-2, -6, and -9) but not to others (e.g., AHSV-5 and -7). Hyperimmune monospecific antisera raised in rabbits with purified VP2 neutralized the infectivity of a virulent strain of AHSV-4 isolated from an infected horse during a recent outbreak of the disease in Spain.

African Horse Sickness↗
PubMed1994

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis.

Immunoblot detection of antibody against 47 KD cytoplasmic antigen of Candida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.

Agglutination Tests↗
PubMed1994

Synthesis of recombinant baculoviruses expressing the outer capsid protein VP2 of five BTV serotypes and the induction of neutralizing antibodies to homologous and heterologous BTV serotypes.

DNA representing RNA segment L2 of 5 different bluetongue virus (BTV) serotypes (BTV-1, -2, -11, -13 and -17) corresponding to the gene that codes for the BTV neutralization antigen VP2, have been inserted individually into baculovirus transfer vectors in lieu of the 5' coding region of the polyhedrin gene of Autographa california nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda cells with wild-type AcNPV DNA in the presence of the derived transfer vector DNAs, polyhedrin-negative recombinant baculoviruses were recovered. When S. frugiperda cells were infected with each of these viruses, protein was made similar in size and antigenic properties to the authentic BTV VP2 protein. To evaluate the biological activities of these proteins, antibodies were raised to them in guinea pigs. The neutralization capabilities of these antisera were tested against homologous and, for two of the higher titered sera, heterologous BTV serotypes. Each antiserum neutralized the infectivity of the homologous virus serotype. In heterologous virus challenges, the serum raised to the VP2 protein of BTV-13 also neutralized the infectivity of BTV-1, and to lesser extents BTV-3, -16, -8 and -9 (BTV-2, -10, -11, -15 were not tested). The serum raised to the VP2 of BTV-17 neutralized BTV-20 and -21, and to lesser extents BTV-4 and -8 (again, BTV-2, -10, -11, -15 were not tested).

Animals↗
PubMed1994

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus.

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%-99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36-50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Amino Acid Sequence↗
PubMed1994

Phylogenetic relationships of the VP2 protein of a virulent isolate of bluetongue virus (BTV-23) compared to those of 6 other BTV serotypes.

To determine the genetic relationship of the virulent Australian bluetongue virus serotype 23 with that of other serotypes and to identify the extent and nature of the antigenic variation among seven serotypes of bluetongue virus (BTV), the complete nucleotide sequence was determined for cDNA clones representing the L2 dsRNA of BTV-23, the gene that codes for the outer capsid neutralization antigen (VP2). The predicted amino acid sequence of the protein was compared with the VP2 sequences of the five USA serotypes (BTV-2, -10, -11, -13 and -17) as well as with an Australian isolate of BTV-1. The comparisons revealed that the VP2 of BTV-23 is most closely related to that of BTV-1, sharing 52% identical and 72% similar sequences, also that the VP2 of the two Australian serotypes are more closely related to that of BTV-2 than to the other four USA serotypes. Only 22% identical sequences are shared by all seven VP2 molecules; however, when homologous substitutions are considered the similarity index was as high as 48%. In addition, the conserved regions that have been identified previously for other VP2 molecules are also conserved in BTV-23.

Amino Acid Sequence↗
PubMed1994

Long-lasting protection of sheep against bluetongue challenge after vaccination with virus-like particles: evidence for homologous and partial heterologous protection.

Insect cells co-infected with appropriate recombinant baculoviruses synthesize double-shelled, virus-like particles (VLPs) with bluetongue virus (BTV) VP2 proteins representing serotype 1 (BTV-1), 2 (BTV-2), 10 (BTV-10), 13 (BTV-13) or 17 (BTV-17) as previously reported for BTV-10 (French, T.J., Marshall, J.J.A. and Roy, P. J. Virol. 1990, 64, 5696-5700). The derived particles were purified and used to vaccine sheep, either as single VLP types (BTV-10, BTV-17) or as a combination of all five serotypes. Control sheep received saline. The virus-neutralizing antibody responses were measured. Depending on the experiment, the sheep were challenged with homologous (BTV-10, -13, -17) or selected heterologous (BTV-4, -11, -16) viruses either after 4 months or 14 months, and the disease, viraemias and clinical reactions monitored. The results indicated that two doses of 10 micrograms of VLPs elicited a long-lasting immune response which protected the sheep against challenge with the homologous virulent virus. In certain cases, partial protection was afforded against challenge by heterologous BTV serotypes.

Animals↗
PubMed1994

Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of nonstructural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells.

Virus-specific tubules are characteristic of orbivirus infections and are likely to play an important role in virus morphogenesis. It has been shown that for bluetongue virus (BTV), the prototype orbivirus in the family Reoviridae, the virus-encoded NS1 protein forms tubules in insect cells when the BTV segment M6 gene is expressed by using a baculovirus vector. To understand the function of NS1 tubules and to identify the sequences involved in their polymerization, a series of mutant NS1 genes was generated and expressed in insect cell cultures by using baculovirus vectors. Three of the mutants were deletion mutants. One (AcNS1.dNT10) lacked 10 of the amino-terminal amino acids, and the other two mutants (AcNS1.dCT20 and AcNS1.dCT43) lacked 20 or 43 of the carboxy-terminal amino acids. In addition, site-directed mutants were constructed in which various single cysteines or pairs of cysteines were changed to serines. The ability of each mutant protein to form tubules was investigated. None of the deletion mutants formed tubules. The constructs in which the cysteines at amino acid positions 337 and/or 340 were replaced by serines (e.g., AcNS1.C337S,C340S) also did not form tubules. Instead, the NS1 protein of these and the deletion mutants made ribbon-like structures which formed large aggregates. Mutations involving six other cysteines (i.e., AcNS1.C37S,C43S,AcNS1.C462S,C465S, AcNS1.C104S, and AcNS1.C364S) produced tubules. The results show that both the amino and carboxy termini of the NS1 protein molecule and the cysteines at residues 337 and 340 are essential for tubule formation.

Animals↗
PubMed1994

Deletion and mutational analyses of bluetongue virus NS2 protein indicate that the amino but not the carboxy terminus of the protein is critical for RNA-protein interactions.

Genome segment 8 (S8) of bluetongue virus serotype 10 (BTV-10) encodes the nonstructural protein NS2. This protein, which has single-stranded RNA (ssRNA) binding capacity, is found in BTV-infected cells in the form of virus inclusion bodies (VIBs). To identify the domain(s) important for RNA binding and oligomerization of the protein, a number of deletions were made in regions of the gene that code for either the amino or carboxy terminus of the protein. The modified genes were cloned into and expressed from baculovirus vectors based on Autographa californica nuclear polyhedrosis virus. Truncated NS2 proteins were individually analyzed for the ability to bind ssRNA and to form VIBs. The results indicated that the carboxy terminus of the protein is involved neither in RNA binding nor in the formation of VIBs. The amino terminus of NS2 was shown to be essential for ssRNA binding but not for NS2 protein oligomerization. Point mutations that involved the substitution of various charged residues at the amino terminus of NS2 were generated and tested for the ability to bind ssRNA. The results showed that the arginines at amino acid residues 6 and 7 and the lysine at residue 4, but not the glutamic acid at residue 2, are involved in ssRNA binding.

Amino Acid Sequence↗
PubMed1994

Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores.

Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation.

Amino Acid Sequence↗
PubMed1994

Infection with fatal outcome after endovascular metallic stent placement.

The authors describe a case in which infection developed in a patient after placement of a metallic endoprosthesis in the iliac artery. The patient died of respiratory failure secondary to sepsis. Imaging studies did not reveal an abscess, aneurysm, or stent occlusion. Stent cultures and hemocultures grew Staphylococcus aureus. As with any foreign body, infectious complications may occur after insertion of metallic stents and should be recognized as soon as possible to initiate appropriate therapy.

Fatal Outcome↗
PubMed1994

Bladder cancer and occupational exposures.

OBJECTIVES: A hospital-based case-referent study was carried out in Lyon with the purpose of generating hypotheses about the role of occupational exposures to 320 compounds in bladder carcinogenesis. METHODS: Job histories were obtained by questionnaire for 116 cases and 232 reference patients with diseases other than cancer (one referent from the same hospital ward and one from another ward of the same hospital per case); the referents were matched for gender, hospital, age, and nationality. Systematic coding of exposures, with a blind analysis of job histories, was carried out by a team of experts in chemistry and occupational health. RESULTS: Significantly elevated odds ratios were observed for exposure to pyrolysis and combustion products [odds ratio (OR) 2.3, 95% confidence interval (95% CI) 1.0-4.0] when the general referents were used and for cutting fluids (OR 2.6, 95% CI 1.2-5.4) when tobacco consumption was adjusted for. The latter was highest among the category consisting of blue-collar and unskilled workers, supervisors, and agricultural workers (OR 4.6 95% CI 2.0-10.6), while the odds ratio for the other category was 0.8 (95% CI 0.3-2.7). An elevated odds ratio for exposure to inks was observed for the women (OR 14.0, 95% CI 1.8-106.5) on the basis of 14 exposed cases, but confounding factors could have been responsible for this result. Odds ratios for several other exposures (rubber: OR 5.7, nitrates: OR 8.2, coke dust: OR 3.5, meat additives: OR 3.8) were also elevated, but not significantly so when based on a small number of exposed cases. CONCLUSION: The observations of this investigation should be tested in future studies, in particular since exposures to agents such as cutting fluids or pyrolysis products are ubiquitous in industrial settings and may present an important public health hazard.

Adult↗
PubMed1994

[Prognostic factors in colorectal adenocarcinoma of Dukes stage B. Study of a series of population].

A study of prognostic factors in Dukes B colorectal adenocarcinoma was performed on a population basis in order to determine subgroups with poor prognosis which could benefit from adjuvant therapy after surgery. The study considered the 746 cases of Dukes B colorectal carcinoma diagnosed during the 1976-1988 period among Côte-d'Or residents (Burgundy, France). The overall 5-year crude survival rate was 55.7%, the corresponding relative survival rate being 73%. In the final Cox model, age, tumour extension to adjacent organs, number of examined lymph nodes and tumour size were significant prognostic factors. The corresponding multivariate relative survival model considered only tumour extension and number of examined lymph nodes as having a prognostic value. The relative risk of death was 2.3 (range: 1.5-3.3) in case of a tumor extension and 2.5 (range: 1.5-4.4) when no lymph node was found compared to surgical samples with at least 6 lymph nodes. These data should be taken into account when conceiving or analyzing future therapeutic trials.

Adenocarcinoma↗