Complete coding sequence of equine inducible nitric oxide synthase.
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Animals↗
Biomedical subjects
R A Hammond
Publications and source records attributed to R A Hammond.
Vaccine-induced antibodies inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis.
Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management.
Animals↗
Endotoxin induction of nitric oxide synthase and cyclooxygenase-2 in equine alveolar macrophages.
OBJECTIVE: To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. PROCEDURE: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde 3-phosphate dehydrogenase. In a second study, cells were incubated with LPS or vehicle for 24 hours. Culture medium was assayed for COX-2 and iNOS activity by determining prostaglandin E2 (PGE2) and total nitrite concentrations, respectively. RESULTS: Lipopolysaccharide induces COX-2 and iNOS mRNA in equine alveolar macrophages. Sequencing revealed that PCR products for COX-2 and iNOS had a high degree of nucleotide homology with the human sequences (91% COX-2, 93% iNOS). Production of mRNA for COX-2 and iNOS was accompanied by induction of enzyme activity. Comparing PCR fragment production, expression of mRNA for iNOS appeared to be less than that for COX-2. Induction of COX-2, but not iNOS, was LPS-concentration dependent. Conclusion-Lipopolysaccharide induces COX-2 and iNOS in equine macrophages. CLINICAL RELEVANCE: The induction of iNOS and COX-2 by LPS in equine macrophages suggests these enzymes may be important in the pathophysiology of sepsis. Pharmacologic modulation of iNOS and COX-2 activity may represent a novel therapeutic target in the management of endotoxemia in horses.
Animals↗
The moral imperatives for diversity.
The debate over diversity and affirmative action remains contentious at best and divisive or polarizing at worst. The profession of orthopaedics, like all elite professions, is very much involved in this debate. Starting with the assumption that there are reasonable arguments on both sides of this question, this paper will search for common ground by focusing on three issues: the ethical and pragmatic reasons for overcoming the present effects of past discrimination; the importance of ensuring the clinical and cultural competence of the specialty of orthopaedics in taking care of a diverse population; and the value of preserving a commitment to equality.
Clinical Competence↗
Surgical management of subvalvular aortic stenosis and mitral dysplasia in a golden retriever.
A 12-month-old neutered male golden retriever was presented with a history of lethargy and exercise intolerance. Clinical examination, electrocardiography, radiography and echocardiography supported a diagnosis of fixed subvalvular aortic stenosis with a Doppler pressure gradient of 77.5 mmHg. Surgical inspection also revealed gross structural abnormalities of the mitral valve consistent with mitral dysplasia. Intervention consisted of resection of the dysplastic mitral valve and the subvalvular aortic stenosis. The mitral valve was replaced with a bioprosthetic valve. Total cardiopulmonary bypass time was 65 minutes and aortic cross-clamp time was 55 minutes. A full recovery was made and 11 months postoperatively the aortic transvalvular gradient was 30 mmHg. At the time of writing, 12 months after surgery, the dog was clinically normal and requires no medication.
Animals↗
Surgical treatment of intrahepatic portosystemic shunts in six cats.
The surgical treatment of intrahepatic portosystemic shunts in six cats is described. The preoperative diagnoses were based on the results of abdominal ultrasonography, and mesenteric portography was used during surgery to confirm the diagnosis and establish the morphology of the shunting vessel. In four of the cats the shunt vessel passed through the left division of the liver, compatible with a patent ductus venosus (PDV), in one cat the shunt passed through the central hepatic division and in the other cat it passed through the right hepatic division. During surgery the shunt vessel was manipulated directly either intrahepatically, or post hepatically in the four cats with PDV. In five cats the shunt vessel was partially ligated, and in the other the vessel was completely ligated. The attenuation procedure produced a mean increase in mesenteric venous pressure of 17 cm H2O, with a range from 14 to 20 cm. All the cats recovered from the surgical procedure, but one developed neurological signs shortly after the operation and died from respiratory failure. Four of the cats were clinically normal and required no medication by one month after the operation. One cat required long-term medication to control its continued clinical signs.
Animals↗
Cardiopulmonary effects of desflurane in the dog during spontaneous and artificial ventilation.
The cardiopulmonary effects of desflurane at end tidal concentrations of 10.3, 12.9 and 15.5 per cent during either spontaneous or artificial ventilation were studied in five beagle dogs. Desflurane anaesthesia resulted in tachycardia and a decrease in arterial blood pressure which were not significantly related to the end tidal desflurane concentrations or the mode of ventilation. At an end tidal desflurane concentration of 15.5 per cent there was a significant increase in central venous and pulmonary arterial wedge pressures and, with artificial ventilation, a reduction in cardiac output and stroke volume when compared with similar measurements at an end tidal desflurane concentration of 10.3 per cent. When allowed to breathe spontaneously, the dogs panted at times when they were lightly anaesthetised, but their respiration was depressed to a varying extent at the highest end tidal desflurane concentration. The induction of anaesthesia with desflurane was smooth, and the quality of anaesthesia during maintenance was excellent. There was one episode of a transient tachyarrhythmia associated with the measurement of cardiac output, but no other side effects were observed.
Anesthesia, General↗
Romifidine as a premedicant to propofol induction and infusion anaesthesia in the dog.
The effects of premedication with four different intravenous doses of romifidine (20, 40, 80 and 120 micrograms/kg bodyweight) and a saline placebo were compared in a group of 20 adult beagles of both sexes, undergoing anaesthesia with propofol for a clinical dental procedure. Anaesthesia was induced 10 minutes after premedication and maintained by intravenous infusion of propofol for a period of 30 minutes. Romifidine had a marked synergistic effect with propofol and reduced the required induction and infusion doses by more than 60 per cent for a standard level of anaesthesia; the synergistic effect was dose related. Following premedication, propofol produced no significant alteration of respiratory rate, heart rate or rectal temperature. Anaesthesia was found to be more stable following romifidine premedication at all doses studied. The quality of induction was unaltered by the dose of the romifidine. Recovery from anaesthesia was smooth and of a similar quality in all cases. There were no differences in the recovery times between the unpremedicated group and the dogs premedicated with any dose of romifidine studied. There were no adverse effects noted following this anaesthetic regimen. The marked dose-related synergism with propofol induction and infusion anaesthesia is relevant should romifidine be used in the dog in clinical veterinary practice.
Anesthesia, Dental↗
Mitral valve replacement for the treatment of congenital mitral dysplasia in a bull terrier.
A 10-month-old entire male bull terrier was presented with a history of collapse on exercise. Clinical examination, electrocardiography, radiography and echocardiography all supported a diagnosis of mitral valve dysplasia. There was no evidence of congestive heart failure. Surgical intervention consisted of open resection of the dysplastic mitral valve and its replacement with a bioprosthetic valve. The procedure was performed through a median sternotomy and the dog was placed on to full cardiopulmonary bypass. Total cardiopulmonary bypass time was 70 minutes. A full recovery was made and at the time of writing (17 months postoperatively) the dog is clincally normal requiring no medication.
Animals↗
Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.
Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.
Amino Acid Sequence↗
Overproduction and purification of Bacillus subtilis DNA polymerase III.
The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.
Bacillus subtilis↗
Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene.
Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.
Alleles↗
Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.
The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.
Antibodies↗
The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III.
Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.
Amino Acid Sequence↗
Association of 3'----5' exodeoxyribonuclease activity with DNA replitase complex from S-phase Chinese hamster embryo fibroblast cells.
DNA replitase has been described as a complex of enzymes/proteins that are associated with both DNA precursor biosynthesis and DNA replication in mammalian cells [Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci. USA 77, 3312-3316]. We demonstrate for the first time a 3'----5' exodeoxyribonuclease activity is associated with the replitase complex. As much as 60% of this exonuclease activity was similar to that associated with DNA polymerase delta based upon its sensitivity to inhibition by GMP and by butyl-phenyl-deoxyguanosine triphosphate (BuPdGTP). Association of 3'----5' exonuclease activity with the DNA polymerase in the replitase complex was also demonstrated by analyzing dTTP turnover to dTMP in an in vitro DNA polymerase assay system. The DNA polymerase activity in replitase complex exhibited a sensitivity to BuPdGTP which both was similar to that of DNA replication in permeable cells and was intermediate between the BuPdGTP inhibition of purified DNA polymerases alpha and delta. These studies suggest that the replitase complex contains 3'----5' exonuclease activity associated with the DNA polymerase activity responsible for nuclear DNA replication in mammalian cells. Further studies are required to determine if these activities are at least partially attributed to DNA polymerase delta.
Animals↗
Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha.
Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase alpha (Pol alpha) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Pol alpha enzymes were associated with discrete, Ca2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Pol alpha holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase.primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Pol alpha enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Pol alpha. Immunopurified preparations of the human and hamster Pol alphas retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150-200 kDa.
Animals↗
Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication.
DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A microinjected monoclonal antibody against human DNA polymerase-alpha inhibits DNA replication in human, hamster, and mouse cell lines.
We have examined the effect that microinjection of a monoclonal antibody directed against human DNA polymerase-alpha (SJK-287) has on DNA synthesis in exponentially growing human, mouse, and hamster cell lines. We show that the SJK-287 antibody, when microinjected directly into the nuclei of cells is capable of inhibiting DNA synthesis in all three cell lines tested. Moreover, the effectiveness with which this antibody can inhibit ongoing DNA synthesis by the microinjection assay is closely correlated with the ability of the antibody to neutralize DNA polymerase-alpha activity fractionated from each cell line in vitro. Two other monoclonal antibodies of the same class, one directed against the cellular p53 protein (PAb122), and one directed against the c-myc protein (PM-8) were also tested for their ability to inhibit ongoing DNA synthesis by direct microinjection and in lysolecithin permeabilized cells. Both monoclonal antibodies failed to inhibit ongoing DNA synthesis in exponentially growing cells by these assays.
Animals↗