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M J Moore

Publications and source records attributed to M J Moore.

At least 19 recordsLinked to original sources
PubMed1992

Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites.

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Animals↗
PubMed1992

C-terminal cysteines of Tn501 mercuric ion reductase.

Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway. A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site. However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials. We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity [Moore, M. J., & Walsh, C. T. (1989) Biochemistry 28, 1183; Miller, S. M., et al. (1989) Biochemistry 28, 1194]. In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine. Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function. Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km. In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca. 10-fold in vitro. From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).

Alanine↗
PubMed1991

Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607.

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.

Bacillus↗
PubMed1991

Sarcoptic mite hypersensitivity and skin lesions in slaughtered pigs.

The association between papular skin lesions in slaughtered pigs and hypersensitivity to Sarcoptes scabiei var suis was examined in experimental and field studies, and by the retrospective analysis of monitoring records obtained at slaughter. A causal role for S scabiei was indicated by the production of lesions in experimentally infested pigs, by increased clinical mange in herds having high lesion scores, and by a reduction in the severity of lesions after the implementation of more intensive mange control measures. The specificity of the lesions as an indicator of sarcoptic mite hypersensitivity was estimated to be 0.92 from experimental data and 0.79 from field data. Generalised lesions were rare in mange free pigs, indicating that such lesions were highly specific (1.0 from experimental data, greater than 0.98 from field data) for mange. The assessment of the severity of the lesions in samples of slaughtered pigs appears to be a useful aid to assessing the severity of sarcoptic mange in pig herds.

Animals↗
PubMed1991

Regeneration of rat extensor digitorum longus tendon: the effect of a sequential partial tenotomy on collagen fibril formation.

Alterations in collagen fibril diameter distribution were measured in rat extensor tendons subsequent to two-successive partial tenotomies, 30 days apart. Subsequent to surgery three areas were created. The primary lesion area, consisted of a regenerating window in the central half of the tissue. Secondary lesion areas consisted of strips at the lateral margin of the tissue, removed during the second operation. Between the lesion areas were the non-lesion areas consisting of tissue not removed during surgery. Tissue was examined 40, 60, 120 and 240 days after the initial operation. Within each group, primary and secondary lesion areas remained unimodal in collagen fibril diameter distribution throughout the period of the study. This distribution was initially unimodal in the non-lesion areas but became multimodal by 240 days postoperatively. Both the primary lesion area and non-lesion areas exhibited an increase in frequency of small diameter fibrils between 40 and 60 days postoperatively. It is suggested that all observations may be related to the level of applied stress.

Animals↗
PubMed1991

Seasonality of sarcoptic mange in pigs in South Australia.

Retrospective analysis of records, from July 1987 to June 1991, of 1140 inspections of slaughtered pigs from 154 swine herds in the Pig Health Monitoring Scheme of South Australia was conducted to evaluate patterns of variation in the prevalence and severity of skin lesions typical of sarcoptic mange. An increase in the severity of sarcoptic mange in winter and spring was observed. There was a substantial reduction in the severity of mange over the period of monitoring. These observations support the role of monitoring at slaughter in the management of endemic disease. They provide some basis for refining control programs for mange to improve cost-effectiveness.

Animals↗
PubMed1991

Clinical pharmacokinetics of cyclophosphamide.

Cyclophosphamide has been in clinical use for the treatment of malignant disease for over 30 years. It remains one of the most useful anticancer agents, and is also widely used for its immunosuppressive properties. Cyclophosphamide is inactive until it undergoes hepatic transformation to form 4-hydroxycyclophosphamide, which then breaks down to form the ultimate alkylating agent, phosphoramide mustard. Sensitive and specific methods are now available for the measurement of cyclophosphamide, its metabolites and its stereoisomers in plasma and urine. The pharmacokinetics of cyclophosphamide have been understood for many years; those of the cytotoxic metabolites have been described more recently. The pharmacokinetics are not significantly altered in the presence of hepatic or renal insufficiency. As activity resides exclusively in the metabolites, whose pharmacokinetics are not predicted by those of the parent compound, correlations between cyclophosphamide pharmacokinetics and pharmacodynamics have not been demonstrated. Cyclophosphamide is used in doses that range from 1.5 to 60 mg/kg/day. A steep dose-response curve exists, and reductions in dose can lead to unfavourable outcomes. Myelosuppression is the dose-limiting toxicity, although in the setting of bone marrow transplantation, escalation beyond that dosage range is limited by cardiac toxicity. Longer term complications of cyclophosphamide therapy include infertility and an increased incidence of second malignancies. Cellular sensitivity to cyclophosphamide is a function of cellular thiol concentration, metabolism by aldehyde dehydrogenases to form inactive metabolites, and the ability of DNA to repair alkylated nucleotides. Whether alteration of these cellular functions will lead to further improvements in clinical outcomes is an area of active investigation.

Aldehyde Dehydrogenase↗
PubMed1991

In vivo effects of anti-idiotype on Plasmodium chabaudi infection in mice.

A polyclonal anti-idiotype was raised in rabbits following immunization with a murine monoclonal antibody which recognized a 250,000 MW antigen of Plasmodium chabaudi-infected erythrocytes. The monoclonal antibody, NIMP M23 (clone 3,) has been shown to protect mice against homologous parasite challenge. Following purification, the anti-idiotype was shown to bind only the immunizing idiotype and to recognize antigen-binding site-associated anti-idiotype. Mice primed with anti-idiotype and challenged with live parasites had an altered course of infection, with significant reduction in their peak parasitaemia levels. Anti-idiotype priming did not induce an antigen-reactive antibody response in vivo but a population of T cells capable of proliferating in vitro to P. chaubaudi-infected red cells was stimulated. These data are discussed in the context of possible idiotypic interaction in murine malaria.

Animals↗
PubMed1991

Collagen fibril morphometry in transected rat extensor tendons.

The regenerative potential of completely transected tendon has been assessed in rat extensor tendon. Collagen fibril diameter distributions were calculated within the gap region of the healing tissue at 10, 30, 60, 120 and 240 days postoperatively. At each age the distribution was found to be unimodal with no indication of any return of bimodality. These results suggest that mechanical stimuli may be a controlling factor in determining fibril diameters and that mature tendon may be unable to reproduce a bimodal collagen fibril diameter distribution subsequent to trauma.

Animals↗
PubMed1990

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding.

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.

Animals↗
PubMed1990

Active site of mercuric reductase resides at the subunit interface and requires Cys135 and Cys140 from one subunit and Cys558 and Cys559 from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation.

Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria. The enzyme is an alpha 2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 10(4)-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.

Base Sequence↗
PubMed1990

Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase.

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.

Amino Acid Sequence↗
PubMed1990

Extensor tendon repair: an animal model which allows immediate post-operative mobilisation.

In adult rabbits, an experimentally transected digital extensor tendon was repaired. The technique employed a tongue, created from the proximal tendon, folded over to bridge the transection and reattached distally. Animals were allowed immediate post-operative mobilisation. Tendon material, 14-120 days post-operatively, was examined ultrastructurally in control and experimental animals from four different zones within the repaired tendon. The operation was well tolerated and adhesion free. Associated with the repair, two types of collagen fibril populations were observed. Firstly, in areas where tendon tissue had been removed, there was a population of fibrils with a narrow range of diameters. Secondly, in areas where tendon tissue was subjected to an increased level of stress per unit cross-sectional area, a population of fibrils with a range of diameters similar to that of controls but with a marked increase in the percentage of small diameter fibrils. The relevance of these observations to human tendon repair is discussed.

Animals↗
PubMed1990

Phase II study of trimetrexate in recurrent anaplastic glioma. National Cancer Institute of Canada Clinical Trials Group Study.

The National Cancer Institute of Canada Clinical Trials Group conducted a phase II trial of trimetrexate given in a daily x 5 intravenous bolus schedule every 3 weeks in patients with measurable recurrent anaplastic glioma and limited prior treatment. There were no responses in 14 evaluable patients. We conclude that trimetrexate, given as described, is not an active agent in this disease.

Adult↗
PubMed1990

Are treatment strategies of urologic oncologists influenced by the opinions of their colleagues?

In a previous study, urologists, radiation oncologists and medical oncologists in Britain, Canada and the United States were asked to state how they would wish to be treated if they had urologic cancer as described in six clinical scenarios and whether they would agree to be entered in randomised clinical trials. This study disclosed major controversy regarding treatment options for each scenario and reluctance by these experts to enter randomised clinical trials. In the present study a second questionnaire which included a summary of the treatments selected initially was sent to the same 227 oncologists. Respondents were asked, in view of these additional information, how they would wish to be treated and whether they would enter themselves (or their patients) on randomised trials comparing the two treatment options most favoured by their colleagues. Most respondents did not modify their treatment preference. There was still poor agreement to enter themselves on trials (29%), but a higher proportion would offer such trials to their patients (45%). Thus the demonstration of controversy about optimum treatment did not influence personal bias, but could facilitate the entry of patients into trials that address major controversies. We conclude that treatment strategies of urologic oncologists are influenced minimally by opinions of their colleagues, but that the method of using surrogate questionnaires is a valuable aid to the design of clinical trials.

Aged↗
PubMed1990

Mutations in c-Ki-ras oncogenes in diseased livers of winter flounder from Boston Harbor.

Livers of a natural population of winter flounder from a contaminated site in Boston Harbor were examined for the presence of oncogenes by transfection of DNA into NIH 3T3 mouse fibroblasts. Tissues analyzed contained histopathologic lesions including abnormal vacuolation, biliary proliferation, and, in many cases, hepatocellular and cholangiocellular carcinomas. Fibroblasts transfected with liver DNA samples from 7 of 13 diseased animals were effective in the induction of subcutaneous sarcomas in nude mice. Further analysis revealed the presence of flounder c-Ki-ras oncogenes in all 10 nude mouse subcutaneous tumors analyzed. Direct DNA sequencing and allele-specific oligonucleotide hybridization following amplification of the tumor DNA by the polymerase chain reaction showed mutations in the 12th codon in this gene. Analysis of DNA from all nude mouse tumors as well as the livers from which they were derived showed mutations at this codon. The mutations comprised G.C----A.T or G.C----T.A base changes resulting in substitution of serine, valine, or cysteine for glycine. Liver DNA samples from five histologically normal livers of animals from a less polluted site were ineffective in the transfection assay and showed only wild-type DNA sequences (GGT) at the 12th codon of c-Ki-ras. The prevalence of mutations in this gene region was associated with the presence of liver lesions and could signify DNA damage resulting from environmental chemical exposure.

Animals↗
PubMed1989

Purification, crystallization, and preliminary x-ray diffraction studies of the flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607.

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.

Amino Acid Sequence↗