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Biomedical subjects

P Roy

Publications and source records attributed to P Roy.

At least 127 records · Page 7Linked to original sources
PubMed1998

[Radiotherapy of cancer of the anal canal. 15 years experience at the Lyon civil hospices and brief review of the literature].

AIM: Retrospective analysis of a series of 287 anal canal carcinomas seen during a 15-year period in the Department of Radiotherapy Oncology of Hospices Civils de Lyon. MATERIAL AND METHOD: Between 1980 and 1995, 287 patients were managed in the radiotherapy department of Hospices Civils de Lyon. In 25 cases, the patients were referred for recurrence. Post or preoperative irradiation was performed in 12 and 23 cases. Palliative treatment was given to 15 cases and simple follow-up in 7 cases. Radiotherapy was given to 205 patients. In 71 cases irradiation alone and in 134 concomitant radiochemotherapy was given. RESULTS: The 5-year overall survival of the group treated by radiotherapy (205 patients) was 71.5% and the 10 year-overall survival was 60.8%. The specific survival at 5 and 10 years was 81.9% and 74.7% respectively. At 5 years the overall survival was 78% for the group treated by concomitant radiochemotherapy and 60% for the group treated by irradiation alone. CONCLUSION: Radiotherapy is the standard treatment for anal canal carcinoma. Radiochemotherapy seems to improve results in advanced cases. The best irradiation of technique has yet to be defined.

Adult↗
PubMed1998

Significance of antibody coated bacteria in neurogenic bladder cases.

A total of 85 urine samples from 63 patients with neurogenic bladder, were subjected for pus cell counting and culture. Fifty nine (69.4%) samples showing significant bacterial growth were tested for the presence of antibody coated bacteria (ACB). One serum sample per patient was collected for the estimation of C-reactive protein. E. coli was the commonest bacterial isolate (45.8%) from the clinical samples followed by Ps. aeruginosa (20.3%) and K. pneumoniae (18.6%). Thirty two culture positive samples belonging to 27 patients, were found to be antibody coated. Of all the isolates, K. pneumoniae was antibody coated in higher proportion (63.7%) followed by Ps. aeruginosa (58.3%) and E. coli (55.6%). Immunoglobulin G was found in all the ACB positive samples, where as 87.5% of them were also coated with IgA class of antibodies. All patients with ACB in urine had raised serum CRP levels except one, where as all the rest of the patients had CRP level within normal limit. The presence of pus cells in urine and radiological abnormalities in the urinary tract were significantly higher in ACB positive patients than in ACB negative patients.

Adolescent↗
PubMed1998

[Reproducibility and prognostic value of histopathological classifications of malignant lymphomas. Prolegomena for the 1st international classification proposed by WHO. Group of the non-Hodgkin's Malignant Lymphoma Classification Project].

Different histopathologic classifications have been used in the study of malignant lymphomas. The clinical relevance (reproducibility, prognostic value) has not been precisely studied. The "non-Hodgkin's lymphoma classification project" has been organized to study a cohort of 1,403 cases in 9 sites around the world consisting of consecutive patients seen between 1988 and 1990 in order to have a good follow-up. The reproducibility of the up-dated Kiel and the ILSG (REAL) classifications between the 5 visiting expert hematopathologists was pretty good, at least 85% for the majority of the entities. According to survival curves, the lymphomas can be stratified in 4 different groups. Comparison with the international prognostic index demonstrate that for therapeutic strategy both histopathology and index should be used. The results bring a good support to the project of the WHO to propose the first international classification of lymphomas based on both forme classifications.

Age Factors↗
PubMed1997

Effects of domain-switching and site-directed mutagenesis on the properties and functions of the VP7 proteins of two orbiviruses.

Based on the crystal structure of the VP7 major core protein of bluetongue virus serotype 10 (BTV-10) and that of the top domain of the VP7 protein of African horsesickness virus serotype 4 (AHSV-4), chimeras and site-directed mutants of the proteins were constructed and the products analyzed with respect to their properties and functions. Chimeras with the central upper domain of BTV-10 VP7 replaced by that of AHSV-4 VP7 (construct BAB) formed trimers, as did the converse construct (ABA). Further, both proteins exhibited the expected conformational epitopes of the constituent sequences. Using BAB VP7 it was demonstrated that residues of the upper domain of AHSV-4 VP7 contribute to the observed insolubility of the protein. By contrast, ABA VP7 protein was as soluble as wild-type BTV-10 VP7. Replacement of selected amino acid residues in the top domain (e.g., A167 by R; F209 by T) improved the solubility of BAB VP7. Since the trimeric BAB and ABA VP7 proteins did not form core-like particles (CLPs) when coexpressed with BTV VP3, it was concluded that trimerization of chimeric VP7 is not sufficient for CLP formation. When the N-terminal region of the ABA protein (aa 1-120) was replaced by the respective sequences of BTV VP7 (construct BBA), the protein aggregated and did not form CLPs with coexpressed BTV VP3, most likely due to disruption of the required contacts between the N- and C-terminal regions of the bottom domain, leading to incorrect folding of the chimera.

Amino Acid Substitution↗
PubMed1997

Structures of orbivirus VP7: implications for the role of this protein in the viral life cycle.

BACKGROUND: Bluetongue virus (BTV) is the prototypical virus of the genus orbivirus in the family Reoviridae and causes an economically important disease in domesticated animals, such as sheep. BTV is larger and more complex than any virus for which comprehensive atomic level structural information is available. Its capsid is made primarily from four structural proteins two of which, VP3 and VP7, form a core which remains intact as the virus penetrates the host cell. Each core particle contains 780 copies of VP7. The architecture of the trimeric VP7 molecule has been revealed by crystallographic analysis and is unlike other viral coat proteins reported to date. RESULTS: Two new crystal structures of VP7 have been solved, one (a cleavage product) at close to atomic resolution and the other at lower resolution. The VP7 subunit consists of two domains. The smaller, 'upper', domain is exposed on the core surface and has the beta jelly-roll motif common to many capsid proteins. The second, 'lower', domain is composed of a bundle of alpha helices. The cleavage product comprises the upper domain, which forms a rigid invariant trimeric fragment. The lower resolution structure of the intact molecule indicates that the alpha-helical domain can rotate about the linker to the upper domain to adopt radically different orientations with respect to the threefold axis in the intact protein. CONCLUSIONS: The crystal structures of VP7 reveal a remarkable mix of rigidity and flexibility that may provide insights towards understanding how VP7 interacts with the other capsid proteins of different stoichiometries. These results suggest that substantial conformational changes in VP7 occur at some stage in the viral life cycle. Such changes may be related to the central role that VP7 is likely to play in cell attachment and membrane penetration.

Bluetongue virus↗
PubMed1997

An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.

Bluetongue virus↗
PubMed1997

A new, rapid and simple procedure for direct cloning of PCR products into baculoviruses.

We propose a novel method for direct cloning of foreign genes into baculoviruses which avoids the use of bacterial transfer vectors. The foreign gene to be inserted is derived by PCR using appropriate primers each of which contains an additional 50 nt of baculovirus sequence for homologous recombination between the PCR-derived DNA and the baculovirus DNA, thus accomplishing insertion of the foreign gene into the baculovirus. The direct cloning of green fluorescent protein and beta-glucuronidase in different baculovirus loci is described. The method is simple and avoids the use of cumbersome techniques associated with enzymatic treatment and DNA purification.

Baculoviridae↗
PubMed1997

Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles.

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 A in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryo-electron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 microg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.

Baculoviridae↗
PubMed1997

An in vitro force measurement assay to study the early mechanical interaction between corneal fibroblasts and collagen matrix.

An in vitro force measurement assay has been developed to quantify the forces exerted by single corneal fibroblasts during the early interaction with a collagen matrix. Corneal fibroblasts were sparsely seeded on top of collagen matrices whose stiffness was predetermined by micromanipulation with calibrated fine glass microneedles. The forces exerted by individual cells were calculated from time-lapse videomicroscopic recordings of the 2-D elastic distortion of the matrix. In additional experiments, the degree of permanent reorganization of the collagen matrices was assessed by lysing the cells with 1% Triton X-100 solution at the end of a 2-hour incubation and recording the subsequent relaxation. The data suggest that a cell can exert comparable centripetal force during either extension of a cell process or partial retraction of an extended pseudopodia. The rates of force associated with pseudopodial extension and partial retraction were 0.180 +/- 0.091 (x 10(-8)) N/min (n = 8 experiments) and 0.213 +/- 0.063 (x 10(-8)) N/min (n = 8 experiments), respectively. Rupture of pseudopodial adhesion associated with cell locomotion causes a release of force on the matrix and a complete recoil of the pseudopodia concerned; a simultaneous release of force on the matrix was also observed at the opposite end of the cell. Lysis of cells resulted in 84 +/- 18% relaxation of the matrix, suggesting that little permanent remodeling of matrix is produced by the actions of isolated migrating cells.

Animals↗
PubMed1997

Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans.

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains of Thiobacillus ferrooxidans. The 1.4-kb fragment hybridized with IST2, an insertion sequence of T. ferrooxidans. The 1.2-kb fragment was cloned and sequenced. The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with ISAE1 of Alcaligenes eutrophus. The IST445 contains three open reading frames which have codon usage almost similar to 56 different coding genes of T. ferrooxidans. In Southern blots of restricted genomic DNAs probed with IST445, each of the several strains of T. ferrooxidans gives a distinctive fingerprint. IST445 is present in the range of 10-20 copies per genome in the four strains studied.

Amino Acid Sequence↗
PubMed1997

Agar-gel-immunodiffusion and counterimmunoelectrophoresis for diagnosis of Newcastle disease.

Chickens were experimentally infected with Newcastle disease virus (NDV) and different organs were collected at point of death. Demonstration of NDV specific antigen in tissue samples were detected by haemagglutination (HA), agar-gel-immunodiffusion (AGID) and counterimmunoelectrophoresis (CIE) tests. Out of 51 samples tested 44, 27 and 37 were positive by HA, AGID and CIE respectively. The usefulness of AGID and CIE in Newcastle disease diagnosis is discussed.

Agar↗
PubMed1997

Postvaccinal immune response to regimens of Newcastle disease vaccination by filter paper sampling technique.

Seven hundred and ten blood samples were collected at random from commercial layers in Tamil Nadu on Whatman filter paper No. 1 instead of the conventional method of serum collection. The birds were subjected to different Newcastle disease (ND) vaccination schedules and samples were collected to study the vaccinal response to ND at field level. Eluates were obtained from sample areas of filter paper using Brij-35 solution [detergent] and subjected to the micro haemagglutination inhibition (HI) test for ND antibodies. The HI titre ranged from less than 2(4) to 2(9). The possible causes of poor immune response to ND vaccinations are discussed.

Agglutination Tests↗
PubMed1997

Methanol-precipitated, detergent-treated Newcastle disease virus as an agar-gel precipitation test antigen.

Methanol-precipitated, detergent-treated (Triton X-100) Newcastle disease virus (NDV) was found suitable as an agar gel precipitation test (AGPT) antigen. One hundred and twenty sera were tested by the haemagglutination-inhibition (HI) test and AGPT against NDV. There was a very significant increase in the proportion of AGPT positive samples with increase in HI titre. Hence AGPT can be recommended as a field based test for seromonitoring following vaccinations against ND where laboratory facilities are inadequate.

Agar↗
PubMed1997

The relationship between D2 receptor occupancy and plasma levels on low dose oral haloperidol: a PET study.

The purpose of this study was to determine the relationship between dopamine D2 receptor occupancy and plasma haloperidol. Twelve patients treated with 1-5 mg/day of haloperidol had their D2 occupancy measured using [11C]-raclopride and positron emission tomography and haloperidol plasma levels measured using gas chromatograph mass spectrophotometer. The patients exhibited haloperidol plasma levels ranging from 0.5 to 5.8 ng/ml and D2 occupancy from 53 to 88%. The D2 occupancy was related to the plasma level as a saturating rectangular hyperbola relationship (r2 = 0.84) and it showed that, on average, 50% D2 occupancy was achieved with 0.51 ng/ml and 80% D2 occupancy with 2.0 ng/ml. Our findings demonstrate that 2-5 mg/day of haloperidol, which usually leads to plasma levels of 1-2 ng/ml, would be expected to induce 60-80% dopamine D2 receptor occupancy. If, as has been claimed, 70% D2 occupancy is adequate for typical neuroleptic response, then the conventional use of > 10 mg/day may have been too high, since 70% occupancy can be achieved in most patients by 2-5 mg/day. On the other hand, if as others have suggested, 8-12 ng/ml of haloperidol is the correct therapeutic window for plasma levels, then the required therapeutic D2 occupancy is closer to 90%, not 70%. The implications of the D2 occupancy findings for the optimal dosing of neuroleptics are discussed.

Administration, Oral↗
PubMed1997

Transiently decreased sialylation of thyrotropin (TSH) in a patient with the euthyroid sick syndrome.

A 60-year old woman was admitted to the medical intensive care unit with respiratory distress and worsening renal function, and was found to have bilateral renal artery occlusion. Aggressive nutrition per nasogastric tube was begun on day 8 of her illness, and she eventually recovered after bilateral renal artery bypass surgery, which was performed on day 15. She developed the euthyroid sick syndrome. Levels of serum TSH, T3, and T4 fell during the first few days of her illness, then all trended to normal levels by day 28. The TSH level declined from 1.6 microU/mL on day 2 to 0.2 microU/mL on day 5, then rose to 4.5 microU/mL on day 10, and was 3.8 microU/mL on day 14. The ratios of free T4/TSH, a crude measure of the bioactivity of TSH, were 1.4, 8.0, 0.16, 0.32, and 1.14 on days 2, 5, 10, 14 and 28, respectively. TSH was immunoaffinity concentrated from serum collected on four dates. The TSH oligosaccharides were enzymatically released, treated with or without neuraminidase, labeled with a fluorescent probe, and analyzed by gel electrophoresis. The TSH oligosaccharides were found to be transiently less sialylated on day 13 as compared to days 2, 4, and 24. Three gel bands representing poorly sialylated oligosaccharides represented a mean of 20.6% of TSH oligosaccharides on days 2, 4, and 24, but represented 33.7% of TSH oligosaccharides on day 13. This is the first report of altered TSH oligosaccharide sialylation in the euthyroid sick syndrome. If confirmed by studies of additional patients, altered TSH sialylation may, in part, explain the altered TSH bioactivity that has been described in the euthyroid sick syndrome.

Chromatography, Affinity↗
PubMed1997

Low sensitivity of counter-current immuno-electrophoresis for serodiagnosis of typhoid fever.

Counter-current immuno-electrophoresis was evaluated as a diagnostic test for the serodiagnosis of typhoid fever with somatic (O), flagellar (H) and capsular polysaccharide (Vi) antigens of Salmonella typhi on the sera of patients who were blood culture positive (confirmed typhoid cases) or had high Widal agglutination titres, > or = 320, (presumptive typhoid cases). Of the 37 sera from confirmed cases, 30% showed positivity with O antigen, 24% with H antigens and 51% with Vi antigen. In patients with a presumptive diagnosis, 45% were positive for O antibody, 27% for flagellar antibody and 52% for Vi antibody. When all three antigens were combined the reactivity to any of the antigens was found to be 59% in confirmed typhoid cases, 79% in presumptive typhoid cases and 93% in patients who were simultaneously positive by blood culture and Widal agglutination. However, none of the sera from 45 controls gave a positive precipitation reaction with any of the antigens. It is concluded that counter-current immuno-electrophoresis is a rapid test with low sensitivity and high specificity with Vi antigen, a panel of antigens being most effective, and is, therefore, recommended for rapid diagnosis of typhoid fever.

Agglutination Tests↗
PubMed1997

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs.

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.

Animals↗