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Biomedical subjects

M V Martin

Publications and source records attributed to M V Martin.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification of a common cytochrome P450 epitope near the conserved heme-binding petide with antibodies raised against recombinant cytochrome P450 family 2 proteins.

The cytochrome P450 (P450) proteins constitute a superfamily of enzymes involved in various oxidations and related activities. Polyclonal antibodies raised against bacterial recombinant human P450s varied in specificity, depending upon the individual rabbits used. Several of the antisera raised against P450s 2C10 and 2E1 recognized a number of P450 family 1, 2, and 3 proteins, and two of the less selective antibodies were used to identify cross-reactive epitopes. P450 2C10 peptides reacting with anti-P450 2E1 and P450 2E1 peptides reacting with anti-P450 2C10 were isolated after electrophoresis/immunoblotting and analyzed by Edman degradation. Several of these were in a region near the highly conserved Cys that is a putative axial ligand to the heme. Peptides corresponding to the most conserved regions in this area were synthesized. Anti-P450 2C10 sera did not recognize 14-mer peptides corresponding to the heme-binding region (2C10 410-423 or 2E1 409-422) or the 14-mer peptides immediately C-terminal to these (2C10 425-438 or 2E1 424-437), but anti-P450 2E1 sera showed weak reaction with the latter two synthetic peptides. A longer peptide (29-mer) of P450 2E1 containing parts of both regions (412-440) reacted with both anti-P450 2C10 and anti-P450 2E1 antisera. Antibodies raised against a conjugate of the 29-mer peptide (with hemocyanin) recognized this antigen, the more C-terminal 14-mer peptides (2C10 425-438 and 2E1 424-437), P450s 2C10 and 2E1, and P450s 1A1, 11A1, and 17A. The 29-mer peptide showed considerable alpha-helix structure as judged by CD spectroscopy, in contrast to any of the 14-mers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization.

Cytochrome P450 (P450) 2D6 is the classic human liver debrisoquine 4-hydroxylase, the first human P450 for which genetic polymorphism was clearly demonstrated. We prepared 11 different constructs of P450 2D6, with modification at the N-terminus, for expression in Escherichia coli with the vector pCW. These varied considerably in levels of expression of apo- and holoprotein, with the best yield being obtained in a system in which much of the N-terminal hydrophobic segment was removed. Production of holoprotein was highly dependent upon the addition of delta-aminolevulinic acid and FeCl3 to cultures, even though heme production should not be limiting in this system. The expressed protein was not tightly bound to the "heavier" membrane fraction but did not appear to behave as a soluble protein either. A purification strategy was developed involving fractional centrifugation, Triton X-114 phase separation, and flavodoxin affinity chromatography, which led to recovery of apparently electrophoretically homogeneous protein in good yield. Purified P450 2D6 had the expected N-terminal amino acid sequence and catalytic activities toward debrisoquine (4-hydroxylation) and bufuralol (1'-hydroxylation). The availability of a ready source of the recombinant protein should facilitate physical as well as functional studies and antibody production for other uses.

Amino Acid Sequence↗
PubMed1995

Prevalence of penicillin resistant bacteria in acute suppurative oral infection.

Pus aspirated from acute suppurative oral infections in 78 patients (age range 13-76 years) yielded a total of 331 bacterial strains consisting of 143 facultative anaerobes (predominantly Streptococcus spp.) and 188 strict anaerobes (predominantly Prevotella spp.). Seventy-five isolates (23%) were resistant to penicillin (MIC > 1 mg/L), 37 (11%) were resistant to ampicillin (MIC > 2 mg/L) and 16 (5%) isolates were resistant to amoxycillin/clavulanic acid (MIC > 2 mg/L). Samples from 43 (55%) of the patients yielded at least one penicillin resistant isolate and within this group 30 samples (73%) contained at least one strain which produced beta-lactamase. A history of antibiotic therapy during the 6 months before enrollment in the study did not influence the isolation of penicillin resistant bacteria. It is concluded that penicillin resistant bacteria are often present in the microflora of acute dental infection.

Acute Disease↗
PubMed1995

Mechanisms of cytochrome P450 1A2-mediated formation of N-hydroxy arylamines and heterocyclic amines and their reaction with guanyl residues.

Amine oxidation reactions are catalyzed by cytochrome P450 (P450) and peroxidase enzymes; both types of enzymes appear to function via aminium radical intermediates. N-Dealkylation is favored over N-oxygenation for secondary and tertiary amines with both kinds of enzymes, but in the peroxidase-like enzymes N-oxygenation is even less favorable because of apparent restriction of the Fe-O complex in the active site. Among the rat liver P450s many of the carcinogenic primary arylamines and heterocyclic amines are N-oxygenated by P450 1A2 to form the N-hydroxy arylamine derivatives. Studies with human liver P450s also indicate that P450 1A2 plays a major role in such reactions, although some arylamines such as 4,4'-methylene-bis (3-chloroaniline) and dapsone are preferentially N-oxygenated by P450 3A4. Caffeine N3-demethylation has been developed as a useful marker of P450 1A2 levels in humans; the knowledge that P450 1A2 is the major phenacetin O-deethylase also allows insight into previous human interaction studies. 2-Ethynylnaphthalene is a useful mechanism-based inactivator of rat and rabbit P450 1A2 but not human P450 1A2 enzymes; the peptides labeled in the enzymes have been identified, along with the region in rat P450 1A2 that is modified with the photoaffinity label 4-azidobiphenyl. Microcrystals of rabbit P450 1A2 have been obtained as a first course to realizing the three-dimensional structures of these enzymes. Evidence is also presented that the major C8-guanyl DNA adducts resulting from these arylamines and heterocyclic amines in DNA may be formed via rearrangement of an initial N7-guanyl-2-arylamine adduct: reaction of N-acetoxy-2-aminofluorene with C8-methylguanine derivatives led to the formation of stable N7-substituted species, and reaction of N-acetoxy-2-aminofluorene with C8-bromoguanine yielded N-(C8-guanosinyl)-2-aminofluorene in a reaction best rationalized by such a mechanism.

Amines↗
PubMed1994

Metabolic activation and carcinogen-DNA adduct detection in human larynx.

Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.

Acetyltransferases↗
PubMed1994

Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme.

A full-length human cytochrome P450 (P450) 1A2 cDNA clone and four derivatives in which the 5'-terminus was modified were inserted into the pCW vector and used to transform Escherichia coli cells. Low levels of expression were seen with most of the constructs but high expression levels (245 nmol membrane-bound P450 recovered per liter culture) were achieved when the N-terminus was MALLLAVFL, as reported earlier by Fisher et al. (C. W. Fisher, D. L. Caudle, C. Martin-Wixtrom, L. C. Quattrochi, R. H. Tukey, M. R. Waterman, and R. W. Estabrook, 1992, FASEB J. 6, 759-764). The expressed human P450 1A2 in bacterial membranes was rapidly denatured to cytochrome P420 in the presence of detergents. This denaturation was blocked by the inhibitory ligand alpha-naphthoflavone (alpha NF, 7,8-benzoflavone). Human P450 1A2 was solubilized using high concentrations of sodium cholate and Triton N-101 and could be purified to near homogeneity in high yield in two steps. alpha NF was included in the buffer in the first step and then removed in the second chromatography step along with the detergent. The purified human P450 1A2 was found to be almost completely in the high spin iron configuration, in contrast to P450 1A2 enzymes isolated from rats and rabbits. The enzyme was catalytically active toward the known substrates 7-ethoxyresorufin and phenacetin. The N-terminal appears to be blocked, as is the case for other P450s we have expressed that contain the sequence MALLLAVFL in E. coli. Previously this human P450 has only been available in limited amounts; the methods presented here should facilitate further biochemical and practical studies on this interesting enzyme.

Amino Acid Sequence↗
PubMed1994

The sensitivity to chlorhexidine and cetyl pyridinium chloride of staphylococci on the hands of dental students and theatre staff exposed to these disinfectants.

The aim of this investigation was to study the possible emergence of resistant isolates of the genus Staphylococcus on the hands of dental personnel who use 'Hibiscrub' (chlorhexidine-detergent preparation) and cetyl pyridinium-coated gloves. Resistance was determined by a rate-of-kill technique. In four dental student groups (first, second, third and fourth years) no microorganisms survived 30 min exposure to cetyl pyridinium chloride (CPC) or to chlorhexidine diacetate (CDA). In a theatre staff group, no microorganisms survived 30 s exposure to CPC; and only one of 23 isolates survived 30 min exposure to CDA, but was killed after 60 min exposure. It is concluded that staphylococci resistant to either of these disinfectants do not present a problem in dental students or theatre staff.

Aerosols↗
PubMed1993

A comparative study of cephradine, amoxycillin and phenoxymethylpenicillin in the treatment of acute dentoalveolar infection.

A comparative double blind trial was undertaken of the efficacy of cephradine, amoxycillin and phenoxymethylpenicillin in the treatment of acute dentoalveolar abscesses. Patients were admitted to the trial with acute dentoalveolar abscesses with systemic involvement and assessments were made of pain, swelling, temperature and lymphadenopathy. After admission to the trial appropriate surgical therapy was undertaken. The patients were then given either phenoxymethylpenicillin 250 mg four times daily, cephradine 500 mg twice daily or amoxycillin 250 mg three times daily, all for 5 days. The patients were assessed after 2 days and 5 days. All the patients recovered with those taking cephradine showing the best recovery in the first two days of treatment.

Actinomyces↗
PubMed1993

Metabolic activation, DNA adducts, and H-ras mutations in human neoplastic and non-neoplastic laryngeal tissue.

Metabolic activation, DNA adducts, and H-ras mutations were examined in human laryngeal tissue (n = 16) from both smoker and non/ex-smoker patients with laryngeal cancer. DNA adducts detected by 32P-postlabelling were evident only in smokers (n = 13); in fact, smoking cessation for as little as 10 months resulted in no DNA adducts detected (n = 3). Total DNA adduct levels in these samples were significantly correlated with levels of cytochromes P-4502C and 1A1 in laryngeal microsomes. Moreover, the P-4501A1 levels represent the highest yet found in human tissues. In contrast, laryngeal microsomes did not have detectable P-4501A2 activity, while laryngeal cytosols showed appreciable N-acetyltransferase activity for p-aminobenzoic acid (NAT1) but not sulfamethazine (NAT2). DNA was extracted from laryngeal specimens and amplified by PCR. Nylon filter dot or slot blots were hybridized with 32P-labelled probes for codons 12, 13, and 61 of the H-ras gene. Sixty percent of specimens demonstrated mutations in either codon 12, 13, or 61; a single common and specific mutation was a Gln-->Glu transversion in codon 61. This mutation appeared in 5 laryngeal specimens, all from smokers. These results implicate cigarette smoke components, bioactivated by CYP1A1 and/or CYP2C, in DNA adduct formation. These results also demonstrate a probable smoking-related H-ras Gln-->Glu transversion in codon 61.

4-Aminobenzoic Acid↗
PubMed1993

Irradiation mucositis: a reappraisal.

Irradiation mucositis is a complication of anticancer therapy. It is regarded as an unavoidable consequence of treatment. Recent studies have shown that this condition is probably a consequence of abnormal Gram negative bacillary carriage in the oral cavity. If this abnormal carriage is avoided then prevention or amelioration of irradiation mucositis may be possible.

Cranial Irradiation↗
PubMed1992

Modification of cytochrome P450 1A2 enzymes by the mechanism-based inactivator 2-ethynylnaphthalene and the photoaffinity label 4-azidobiphenyl.

2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1992

Amoxycillin-resistant oral streptococci and experimental infective endocarditis in the rabbit.

The ability of three amoxycillin-resistant strains of Streptococcus sanguis 254, 24 and 297 (MIC 40 mg/L) to cause infective endocarditis (IE) in the rabbit was investigated. These strains all produced infection in the rabbit, as did an antibiotic sensitive control strain NCTC 7864. Prophylactic amoxycillin (400 mg/kg body weight) administered one hour before bacterial challenge prevented 80% of the animals developing IE irrespective of the challenging strain. It is concluded that amoxycillin-resistant strains of S. sanguis can cause IE and that amoxycillin prophylaxis can still be effective against these bacteria.

Amoxicillin↗
PubMed1992

A longitudinal study of the effects on the oral mucosa of treatment for acute childhood leukaemia.

A longitudinal study was undertaken of the effect of therapy on the oral mucosa of 12 children with acute leukaemia. The major oral problem was ulceration, which was associated with neutrophil counts of less than 1.0 x 10(9)/l. Candida carriage rates were consistently high in patients with or without oral candidosis. Salivary flow was not significantly different between leukaemic children and the control group.

Antineoplastic Agents↗