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R S Roy

Publications and source records attributed to R S Roy.

At least 19 recordsLinked to original sources
PubMed2004

Conformational properties of hybrid peptides containing alpha- and omega-amino acids.

This review briefly surveys the conformational properties of guest omega-amino acid residues when incorporated into host alpha-peptide sequences. The results presented focus primarily on the use of beta- and gamma-residues in alphaomega sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between alpha-peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and beta-hairpin conformations permits the characterization of backbone conformational parameters for beta- and gamma-residues inserted into regular alpha-polypeptide structures. Substituted beta- and gamma-residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral beta,beta-disubstituted gamma-amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the Cbeta-Cgamma (theta1) and Calpha-Cbeta (theta2) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.

Amino Acids↗
PubMed1999

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17.

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.

Amino Acid Sequence↗
PubMed1999

Expressed protein ligation to probe regiospecificity of heterocyclization in the peptide antibiotic microcin B17.

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains thiazole and oxazole heterocycles derived from a distributive yet directional cyclization of cysteines and serines in the McbA precursor catalyzed by MccB17 synthetase. Whether the formation of upstream rings potentiates downstream heterocyclization has not been previously determined. RESULTS: McbA fragments (46-61 residues) containing glycine substitutions or homocysteine at select upstream cysteine or serine sites were assembled using expressed protein ligation (EPL). Most of these substrates were only partially cyclized by MccB17 synthetase, in contrast to the efficient processing of wild-type McbA(1-61). Homocysteine was not processed to the six-membered heterocycle. CONCLUSIONS: The formation of upstream rings in McbA potentiates the cyclization of carboxy-terminal cysteines and serines, probably by selecting against unfavorable substrate conformations. EPL allows structure-function analysis including unnatural amino acid placements to probe the regiospecificity and chemoselectivity of post-translational heterocyclization during antibiotic maturation.

Amino Acid Sequence↗
PubMed1999

Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. gallinarum, M. iners, M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 10(5) colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.

Animals↗
PubMed1998

Kinetics and regioselectivity of peptide-to-heterocycle conversions by microcin B17 synthetase.

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings introduced post-translationally in the 69 amino acid McbA gene product, an MccB17 precursor, by the microcin B,C,D enzyme complex. Both monocyclic and 4,2-bis-heterocyclic moieties are generated. The enzymatic cyclization involves 14 of the last 43 amino acids of McbA and requires the presence of the first 26 amino acids that function as a specificity-conferring propeptide. RESULTS: We have constructed maltose-binding protein (MBP)-McbA1-46 fusion proteins and have mutagenized the Gly39-Ser40-Cys41 (GSC) wild-type sequence to assess the regioselectivity and chemoselectivity of MccB17-synthetase-mediated heterocycle formation at the first two loci, residues 40 and 41 of McbA. Four single-site and four double-site substrates showed substantial differences in turnover as assessed by western assays, UV-visible spectroscopy and mass spectrometry. Cysteine-derived thiazoles form at a greater rate than serine-derived oxazoles. Formation of bis-heterocycles is sensitive both to composition and sequence context. CONCLUSIONS: The E. coli McbB,C,D MccB17 synthetase is the first peptide heterocyclization enzyme to be characterized. This study reveals substantial regioselectivity and chemoselectivity (thiazole > oxazole) at the most amino-terminal bis-heterocyclization site of McbA. The heterocyclization of GSS and GCC mutants of McbA1-46 by MccB17 synthetase demonstrates that the complex can efficiently generate tandem bis-oxazoles and bis-thiazoles, moieties not found in MccB17 but present in natural products such as hennoxazole and bleomycin. The observations suggest a common enzymatic mechanism for the formation of peptide-derived heterocyclic natural products.

Anti-Bacterial Agents↗
PubMed1998

Role of the microcin B17 propeptide in substrate recognition: solution structure and mutational analysis of McbA1-26.

BACKGROUND: The peptide antibiotic microcin B17 (MccB17) contains oxazole and thiazole heterocycles formed by the post-translational modification of four cysteine and four serine residues. An amino-terminal propeptide targets the 69 amino acid precursor of MccB17 (preproMccB17) to the heterocyclization enzyme MccB17 synthetase. The mode of synthetase recognition has been unclear, because there has been limited structural information available on the MccB17 propeptide to date. RESULTS: The solution structure of the MccB17 propeptide (McbA1-26), determined using nuclear magnetic resonance, reveals that McbA1-26 is an amphipathic alpha helix. Mutational analysis of 13 propeptide residues showed that Phe8 and Leu12 are essential residues for MccB17 synthetase recognition. A domain of the propeptide was putatively identified as the region that interacts with the synthetase. CONCLUSIONS: MccB17 synthetase recognizes key hydrophobic residues within a helical propeptide, allowing the selective heterocyclization of downstream cysteine and serine residues in preproMccB17. The determination of the solution structure of the propeptide should facilitate the investigation of other functions of the propeptide, including a potential role in antibiotic secretion.

Anti-Bacterial Agents↗
PubMed1997

Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics.

The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam'. The chimeric Pam residue integrates the heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position. The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme structure/ function relationships in a native-like peptidyl architecture.

Alanine↗
PubMed1990

Monoclonal antibodies to Quebec strain (Q17) of bovine rotavirus.

Fourteen monoclonal antibodies (MAbs) against "Quebec" strain (Q17) of bovine rotavirus were isolated and characterized. Four were specific for viral protein Vp7 and ten were specific for viral protein Vp6. Five different isotypes were represented by this group of antibodies. All of the anti-Vp6 and none of the anti-Vp7 antibodies were sensitive to the effects of periodate on their antigen. The antibodies could be separated into three groups based on their relative resistance to the dissociation of their antigen-antibody complex by thiocyanate. The MAbs cross-reacted with the proteins of porcine and human rotaviruses both by immunoprecipitation and immunoblot analyses. These techniques revealed the differences in Mw of the viral proteins from different serotypes.

Animals↗
PubMed1990

Study of virus excretion in feces of diarrheic and asymptomatic calves infected with rotavirus.

Infection of calves by rotavirus could lead to diarrhea or subclinical disease. Therein, kinetic of viral excretion from a group of asymptomatic calves, previously exposed to a virulent strain of rotavirus, is compared to that of a group the calves of which had diarrhea associated with rotavirus infection. As determined by an enzyme-linked immunosorbent assay (ELISA), duration of rotavirus shedding in feces and maximum yield of virus antigen were similar within the two groups of calves. Rotaviruses isolated from the two above groups of animals were antigenically related as shown by neutralization tests, and showed a similar RNA electrophoretic profile. In conclusion, it is likely that both asymptomatic and diarrheic calves infected by rotavirus are equally a major source of contamination to contact healthy calves.

Animals↗
PubMed1988

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus.

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.

Animals↗
PubMed1988

Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes.

The efficacy of an adjuvanted bovine rotavirus vaccine in pregnant cattle (15 heifers and 2 cows) was studied. Each of 4 animals was inoculated IM at 8, 5, and 2 weeks before parturition with a water-in-oil emulsion containing live purified bovine rotavirus, mineral oil, and a mannide oleate compound. Four other animals were treated identically, except that muramyl dipeptide was added to the virus preparation. Five additional animals were inoculated orally at the same time intervals with adjuvant-free viral suspension, and 4 other pregnant animals inoculated only with buffer served as uninoculated controls. Kinetic studies of the specific immune responses were determined by quantification of the rotavirus-neutralizing antibodies and by a rotavirus lymphocyte stimulation test in vitro. Results showed that only the emulsions induced marked enhancement of rotavirus antibody titers in the serum, colostrum, and milk of inoculated cows. Colostral and milk lymphocytes isolated from these cows had a positive in vitro proliferative response to rotavirus stimulation, which lasted at least 21 days after parturition. The values of the stimulation index obtained with the colostral/milk lymphocytes were higher than those of the blood lymphocytes, reflecting increased lymphocyte activity in the colostrum/milk. However, addition of muramyl dipeptide to the emulsion preparation did not exert any potentiating effect on the immune response to rotavirus. Calves fed for the first 5 days after birth with a rotavirus-immune cell-free colostrum supplement were protected from a rotavirus challenge exposure on the third day after birth. Virus was not detectable in their feces.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1986

Comparison by plaque assay of bovine rotavirus and a bovine enterovirus-like agent.

Enterovirus-like particles from feces of calves are a frequent source of contamination of bovine rotavirus isolates. A study of plaque formation using BSC-1 cells indicated differences in behaviour of the viruses which could be used for differentiation the purification. The enterovirus-like particles produced well-defined plaques earlier and reached their optimal size much more rapidly than did the rotavirus. Furthermore, plaques produced by bovine enterovirus-like particles were significantly larger than those of bovine rotavirus. The viral cytopathic effects on the cells within the plaques were also characteristic for each virus.

Animals↗
PubMed1986

Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds.

Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified.

Animals↗
PubMed1986

Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec.

Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates.

Animals↗
PubMed1985

Free radicals and myocardial ischemia. The role of xanthine oxidase.

Recent studies have established a major role for oxygen-derived free radicals in post ischemic tissue injury to the intestine. During ischemia, there appears to be a calcium-triggered, protease-dependent conversion of the native xanthine dehydrogenase to a superoxide-producing xanthine oxidase. The catabolic degradation of ATP during ischemia provides an oxidizable substrate, hypoxanthine. On reperfusion, molecular oxygen is resupplied and a burst of superoxide production ensues, resulting in extensive tissue damage. The same mechanism appears to occur in myocardial ischemia. Xanthine dehydrogenase rapidly converts to the oxidase during nonperfusion in the rat heart. In the isolated perfused working rat heart model, 40 min of anoxia followed by reoxygenation results in substantial release of creatine kinase. The release of creatine kinase is blocked almost completely by pretreatment of the rats with allopurinol, a specific inhibitor of xanthine oxidase.

Allopurinol↗
PubMed1985

Distinct rotaviruses isolated from asymptomatic calves.

Rotaviruses were isolated following cell culture of the intestinal contents of four non-diarrheic calves. The four isolates were serially propagated in MDBK and BSC-1 cells in the presence of trypsin and produced rotavirus particles morphologically similar to those found associated with diarrhea. They were antigenically related to the Nebraska calf rotavirus (Norden strain) as investigated by immunofluorescence. Three isolates could be distinguished from the reference Nebraska rotavirus by their thermal stability and/or their differential responses to intestinal neutralizing antibodies. Two isolates produced on BSC-1 cells plaques significantly different in size from the reference strain, No significant genomic variations were detected among the isolates.

Animals↗
PubMed1984

Comparative study of bovine rotavirus isolates by plaque assay.

Rotaviruses were isolated on BSC-1 cells from counterimmunoelectrophoresis and/or electron microscopy positive intestinal contents from two asymptomatic and six diarrheic calves from Quebec. The plaque assay was performed using these lines and agar overlay medium containing trypsin and DEAE-dextran. This assay was used to compare the Quebec isolates to an attenuated American strain (NCDV) and another strain (TH) obtained from France. The NCDV strain produced plaques that were significantly larger than those produced by the TH strain. Three Quebec isolates produced plaques similar in size to TH strain, one isolate was similar to NCDV strain and another isolate produced larger plaques than those of both NCDV and TH strains. The other isolates induced the production of plaques that were not significantly different from those of NCDV or TH strains.

Animals↗