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D C Kelly

Publications and source records attributed to D C Kelly.

At least 19 recordsLinked to original sources
PubMed2001

Nitric oxide modulates articular sensory discharge and responsiveness to bradykinin in normal and arthritic rats in vivo.

Nitric oxide is implicated in peripheral nociceptive processing. This study determined the effects of the nitric oxide synthase inhibitor, L-NAME, on neural discharge from articular C-fibre afferents innervating normal and arthritic ankle joints in anaesthetized rats. Intra-arterial injection of L-NAME (10-20 mg kg(-1)) increased neural discharge in normal and arthritic ankle joints, whereas D-NAME (30 mg kg(-1)) had no effect. The excitation induced by L-NAME (20 mg kg(-1)) was reduced by co-injecting the nitric oxide precursor, L-arginine (50 mg kg(-1)). L-NAME (20 mg kg(-1)) also enhanced responsiveness to bradykinin (10 microg) but only in arthritic rats, whereas L-arginine (50 mg kg(-1)) reduced the excitation by bradykinin (30 microg) in both groups. These results provide evidence that nitric oxide modulates articular C-fibre activity and reduces responsiveness to bradykinin.

Afferent Pathways↗
PubMed1996

Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.

Alleles↗
PubMed1994

Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens.

Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C. The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

Bacterial Toxins↗
PubMed1994

Efficient generation of a reshaped human mAb specific for the alpha toxin of Clostridium perfringens.

We have used the technique of antibody reshaping to produce a humanized antibody specific for the alpha toxin of Clostridium perfringens. The starting antibody was from a mouse hybridoma from which variable (V) region nucleotide sequences were determined. The complementarity-determining regions (CDRs) from these V regions were then inserted into human heavy and light chain V region genes with human constant region gene fragments subsequently added. The insertion of CDRs alone into human frameworks did not produce a functional reshaped antibody and modifications to the V region framework were required. With minor framework modifications, the affinity of the original murine mAb was restored and even exceeded. Where affinity was increased, an altered binding profile to overlapping peptides was observed. Computer modelling of the reshaped heavy chain V regions suggested that amino acids adjacent to CDRs can either contribute to, or distort, CDR loop conformation and must be adjusted to achieve high binding affinity.

Amino Acid Sequence↗
PubMed1993

Polymerase chain reaction for detection of herpesvirus simiae (B virus) in clinical specimens.

A polymerase chain reaction (PCR) was designed which is specific to Macaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys.

Animals↗
PubMed1992

Nucleotide sequence analysis of a homologue of herpes simplex virus type 1 gene US9 found in the genome of simian herpes B virus.

The 10K gene of simian herpes B virus (SHBV) has been located and the nucleotide sequence determined. Its relationship to homologous genes in other herpes-viruses has been examined. The SHBV 10K gene exhibits a closer relationship to its homologue in HSV-1 than to those in the other viruses studied. Nucleotide sequence identity of 61% was found between the HSV-1 and SHBV 10K genes, and 57% identity was found between the corresponding predicted protein sequences. A comparison of the amino acid sequences of the herpesvirus 10K proteins has revealed a number of conserved features. These are examined in relation to possible functions of the 10K proteins. Implications for evolutionary relationships between SHBV and other herpesviruses are discussed.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence analysis of genes encoding glycoproteins D and J in simian herpes B virus.

The gene encoding glycoprotein D (gD) of simian herpes B virus (SHBV) was identified by hybridization with the gD gene of herpes simplex virus type 1 (HSV-1). The gene probe bound to a 2.6 kbp SalI-EcoRI fragment of SHBV DNA, which was cloned into a plasmid vector. The nucleotide sequence of the SHBV DNA fragment was determined. Two complete and one partial open reading frames (ORFs) were found. The nucleotide sequences of the two complete ORFs are 57% and 69% identical to HSV-1 genes US5 (encoding gJ) and US6 (encoding gD), respectively. The partial ORF showed 64% similarity with HSV-1 US7 (encoding gI). The SHBV gD gene revealed many features which are also found in the gD homologues of other herpesviruses. The positions of cysteine residues and receptor-binding sites for the predicted protein are shown to be highly conserved.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and physical mapping of the genome of simian herpes B virus and comparison of genome organization with that of herpes simplex virus type 1.

The molecular structure of the genome of simian herpes B virus (SHBV) was determined by restriction endonuclease mapping studies. Genomic DNA was cleaved with restriction endonucleases BamHI and SalI into 41 and 58 fragments, respectively. Most of these fragments were cloned into the plasmid vector pACYC184; uncloned fragments were identified following isolation from agarose gels. Terminal fragments were identified by exonuclease digestion and radioactive end-labelling, and linkage of fragments was deduced by a combination of single and double digest experiments and cross-blot hybridizations. The genome is larger than that of herpes simplex virus type 1 (HSV-1), being approximately 165 kilobase pairs. Like that of HSV-1, the SHBV genome is composed of a long and a short unique region each flanked by inverted repeat sequences, which allow the unique regions to invert relative to one another, resulting in four possible isomeric arrangements of the molecule. Genome locations of several SHBV genes were compared with their HSV-1 homologues.

Animals↗
PubMed1992

Cloning and nucleotide sequencing of the Clostridium perfringens epsilon-toxin gene and its expression in Escherichia coli.

The sequence of 20 amino acids from the N terminus of Clostridium perfringens epsilon-toxin was determined. Some differences between this sequence and the previously published sequence (A. S. Bhown and A. F. S. A. Habeeb, Biochem. Biophys. Res. Commun. 78:889-896, 1977) were found. A degenerate 23-bp pair oligonucleotide probe was designed from the amino acid sequence data and used to isolate a DNA fragment containing the gene encoding epsilon-toxin (etx) from C. perfringens type B. The gene encoded a protein with a molecular weight of 32,981. Upstream of the gene, promoter sequences which resembled the Escherichia coli sigma 70 consensus sequences were identified. The gene was expressed in E. coli, and the cloned gene product reacted with epsilon-toxin-specific monoclonal antibodies and had a molecular weight and isoelectric point similar to those of the native protein. Downstream of etx, two overlapping open reading frames were identified. Each encoded part of a protein which was homologous to the transposase from Staphylococcus aureus transposon Tn4001. Southern hybridization experiments indicated that the etx gene was found only in C. perfringens types B and D, the types which produce epsilon-toxin.

Amino Acid Sequence↗
PubMed1991

Epitope mapping of the alpha-toxin of Clostridium perfringens.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.

Amino Acid Sequence↗
PubMed1991

Production and characterization of monoclonal antibodies directed against the lipopolysaccharide of Francisella tularensis.

Two monoclonal antibodies (FT14 and FT2F11) directed against the lipopolysaccharide (LPS) of Francisella tularensis were produced for use in tests to detect the organism in environmental samples and clinical specimens. The specificity of the antibodies was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Both antibodies detected LPS from F. tularensis by ELISA, but only one antibody, FT14, was serologically active in an immunoblot. Treatment of the LPS with detergents prior to ELISA eliminated its binding to FT2F11 but not FT14. Qualitatively, both antibodies detected 10 different strains of F. tularensis by ELISA, but quantitatively, FT14 gave a detectable reaction with 10(3) organisms, whereas FT2F11 was able to detect only 10(5) organisms. FT14 did not cross-react with LPS from a range of other gram-negative species of bacteria, whereas FT2F11 cross-reacted against Vibrio cholerae LPS. Neither antibody showed cross-reactions when entire gram-negative organisms were used as antigens. In a competition ELISA, the two monoclonal antibodies were shown to compete for different epitopes. FT14 was strongly inhibited by purified O side chain from F. tularensis LPS, but FT2F11 was only weakly inhibited. It was inferred from those results that FT14 is directed against the O side chain and that FT2F11 is directed against the core.

Antibodies, Bacterial↗
PubMed1990

Anti-idiotypic antibody-induced protection against Clostridium perfringens type D.

A monoclonal antibody (BALB/c mouse) with specificity for a neutralizing epitope on the epsilon-toxin produced by Clostridium perfringens type D was used to raise anti-idiotypic antibodies (anti-Id) in different strains of mice and rabbits. These were purified and used in cross-immunization studies to induce anti-(anti-idiotype). All strains of mice and rabbits immunized with BALB/c-derived anti-Id showed a high-titer antibody response directed towards the active site of the toxin. This protected the animals against toxin challenge and against an oral dose of the vegetative organisms. Animals immunized with other anti-Id preparations showed no specific antibody response and were not protected. Guinea pig peritoneal macrophages have a cell surface receptor for the toxin, and incubation of these cells with BALB/c anti-Id allowed them to survive toxin challenge, indicating that occupation of the receptors by the anti-Id prevented binding by the toxin. In conclusion, we have shown that an internal-image anti-Id preparation will induce protective immunity in syngeneic and xenogeneic animals and furthermore that immunity to a single epitope on the exotoxin is sufficient to protect against the toxin and clinical sequelae evoked by the disease-causing organism itself.

Animals↗
PubMed1989

Discrimination between twenty isolates of herpesvirus simiae (B virus) by restriction enzyme analysis of the viral genome.

Twenty isolates, obtained from adult breeding monkeys, were all identified as herpesvirus simiae (B virus) by neutralisation with polyclonal B virus antiserum. Subsequent analysis of restriction enzyme profiles produced by digestion of DNA from the isolates enabled discrimination to be made between them. In particular Cynomolgus monkey isolates could be distinguished from those of Rhesus animals. One isolate (isolate 9) could not be typed either as B virus or as the antigenically related herpesvirus SA8, despite neutralisation by B virus antiserum. Unlike herpes simplex virus, B virus isolates could not be divided into oral and genital types on the basis of restriction enzyme profiles.

Animals↗
PubMed1989

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1985

Neutralisation of Trichoplusia ni nuclear polyhedrosis virus with antisera to two forms of the virus.

Two forms of a baculovirus, Trichoplusia ni nuclear polyhedrosis virus, one released at the plasma membrane of cells (cell released virus: CRV) and the other derived from polyhedra (polyhedron derived virus: PDV) occur naturally. Antisera raised against the two forms specifically neutralised the homologous form. The envelope proteins of the two forms were shown to be the major proteins involved in serum neutralisation and to be responsible for the antigenic diversity of the virus forms.

Animals↗
PubMed1984

Baculovirus replication: effects of inhibitors of macromolecular synthesis.

A number of inhibitors of DNA, RNA, protein and polyamine synthesis have been used to elucidate the mode of replication of baculoviruses. An overview of the viruses, the antimicrobial inhibitors used, and the effects of the drugs are presented. Although certain inhibitors of protein synthesis and DNA synthesis are useful in determining the program of expression of the viral genome into immediate early, delayed early, late and very late phases of synthesis, few drugs have proved useful as antimicrobial agents. Bromovinyldeoxyuridine (BVDU) is a potent inhibitor of baculovirus replication inhibiting viral DNA synthesis by blocking the virus induced DNA polymerase. Preliminary experiments suggest that BVDU suppresses baculovirus disease in insect larvae.

Antiviral Agents↗