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Biomedical subjects

C J Smith

Publications and source records attributed to C J Smith.

At least 127 records · Page 7Linked to original sources
PubMed1996

Loss of nitric oxide production in the coronary circulation after the development of dilated cardiomyopathy: a specific defect in the neural regulation of coronary blood flow.

1. The aims of our study were to determine the role of nitric oxide (NO) in cholinergic reflex dilation of the coronary circulation in normal healthy conscious dogs and after the development of pacing-induced dilated cardiac myopathy and overt congestive heart failure. 2. Dogs were instrumented using sterile surgical techniques under general anaesthesia and allowed to fully recover. The Bezold-Jarisch reflex was stimulated by the intra-atrial injection of veratrine or the intravenous injection of PGI2, while the carotid chemoreflex was stimulated by the intracarotid injection of nicotine. Experiments were performed before and after the development of overt congestive heart failure (HF) caused by rapid left ventricular pacing for 4 weeks. 3. The release of NO, or NO-mediated vascular relaxation following administration of acetylcholine (ACh) may have little physiological significance since as ACh is released from nerve endings in vivo. Stimulation of the Bezold-Jarisch or carotid chemoreflex resulted in typical vagal cholinergic reflex coronary vasodilation, an increase in coronary blood flow and a decrease in coronary vascular resistance, which was substantially reduced following NO synthesis inhibition with nitro-L-arginine. 4. After the development of severe congestive HF, the production of NO by sieved coronary microvessels from the heart was markedly reduced accompanied by a 60-80% reduction in both the mRNA (northern blot) and protein (western blot) for endothelial NO synthase in the aorta. 5. After the development of severe pacing-induced HF, activation of the Bezold-Jarisch or carotid chemoreflex no longer resulted in coronary vasodilation due to the disappearance of NO production from the coronary circulation. 6. Therefore, cholinergic reflex coronary vasodilation is mediated by NO. Because coronary blood vessels lose the ability to produce NO after the development of HF, reflex cholinergic coronary vasodilation is markedly altered, uncovering a previously undiscovered specific defect in the integrated control of the coronary circulation in the failing heart.

Animals↗
PubMed1996

Oral white lesions with special reference to precancerous and tobacco- related lesions: conclusions of an international symposium held in Uppsala, Sweden, May 18-21 1994. International Collaborative Group on Oral White Lesions.

An international group of epidemiologists, clinicians and pathologists with a special interest in oral white lesions and their precancerous significance has reviewed earlier work on this topic and identified some of the problems associated with previous definitions, descriptions and classifications. Modifications to these definitions, descriptions and classifications have been proposed, accompanied by explanations of the reasons for identifying the need for changes to be made. Leukoplakia may be a provisional or definitive diagnosis dependent upon the circumstances of oral examination and the availability of other information. Guidelines are provided to assist in the application of the definitions of oral leukoplakia and illustrations depict the homogeneous and non-homogeneous clinical variants. Consideration is also given to the importance of a red component in a white lesion, or a lesion that is entirely red (erythroplakia). A new clinical staging procedure for oral leukoplakia is also proposed.

Erythroplasia↗
PubMed1996

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.

2,2'-Dipyridyl↗
PubMed1996

Reduced gene expression of vascular endothelial NO synthase and cyclooxygenase-1 in heart failure.

Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures > 25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with 32P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66 +/- 0.77 (mean +/- SEM, n = 17) and 1.12 +/- 0.37 (n = 6 and the ratios of COX-1 to GAPDH were 1.52 +/- 0.52 and 0.56 +/- 0.15 before and after heart failure, respectively. These represent 56% to 64% (P < .05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (P < .05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.

Animals↗
PubMed1996

Correlation of hematologic markers of inflammation and lung function: a comparison of asymptomatic smokers and nonsmokers.

Increased inflammation of the peripheral airways has been implicated as a cause of pulmonary function impairment. However, little information is available on the correlation between subclinical decrements of pulmonary function and inflammation in asymptomatic individuals. A relationship between markers of inflammation and lung function may be useful in predicting the early onset of lung function impairment. The purpose of this study was to investigate the correlation of hematologic markers of inflammation and spirometry in asymptomatic smokers and nonsmokers. The specific objectives of this study were twofold. The first objective was to quantify and compare the spirometric measures of lung function in smokers and nonsmokers having similar demographic and lifestyle characteristics. The second objective was to define the correlation between these spirometric measurements and hematologic markers of inflammation (white blood cells, monocytes, basophils, PGE1, IgG, and IgE). Systemic blood samples and spirometric measurements were obtained from 61 age-matched (33 +/- 9 years) healthy, asymptomatic smokers and nonsmokers, with similar self reported lifestyles (i.e., food, alcohol, vitamin consumption and exercise). Both male and female smokers self reported a higher coffee consumption (P < 0.05) compared to nonsmokers. Male smokers self-reported a trend toward current blue-collar versus white-collar occupation when compared with the nonsmokers. Body weight (77.6 +/- 16.6 kg) did not differ between the smokers and nonsmokers. The male nonsmokers were taller than the male smokers (P < 0.05). All subjects were asymptomatic and had clinically normal spirometry. Compared to male nonsmokers, the male smokers had lower FEF25-75% and FEF75-45% values (P < 0.05). No additional spirometric measurements, including FEV1/FVC, FEV1 and FVC were significantly different. The female smokers did not differ from the female nonsmokers (P < 0.05) in any of the spirometric endpoints measured. Thirteen statistically significant (P < 0.05) correlations involving inflammatory (white blood cells, monocytes, basophils, and PGE1) or immunologic endpoints (IgE) and spirometric measurements were observed in female smokers, female nonsmokers and male nonsmokers. No statistically significant correlations involving immunologic or inflammatory endpoints were observed in the male smokers. A better mechanistic understanding of the observed relationship between elevated hematologic inflammatory endpoints and reduced lung function may provide valuable insight into the clinical significance of these correlations.

Adult↗
PubMed1996

Determination of prothrombin activation fragments in young patients with inflammatory bowel disease.

OBJECTIVE: To assess coagulation cascade activation as a potential index of thromboembolic risk in inflammatory bowel disease (IBD). STUDY DESIGN: Fifty plasma samples were obtained during consecutive outpatient encounters with 29 patients (male:female = 20:9) with either Crohn's disease (CD) (n = 23) or ulcerative colitis (UC) (n = 6). Disease activity for CD was determined using the Pediatric Crohn's Disease Activity Index (PCDAI) and for UC using signs/symptoms and mucosal histology. Patients were grouped as active, recently active (not currently active but had been active within the previous 2 months), or inactive. Prothrombin fragment 1.2 (F1.2) was determined by enzyme-linked immunosorbent assay on plasma samples. Lab parameters were compared using the Kruskal-Wallis test with pairwise comparisons made using Wilcoxian rank sum test. RESULTS: Forty-three percent of samples taken during active phases of disease (13 of 30) had elevated prothrombin cleavage byproduct F1.2. Similarly, five of eight encounters (63%) with recently active IBD had elevated F1.2 values. In contrast, none of the patients with inactive disease exhibited F1.2 elevation. Median F1.2 levels in both the active (0.85 nM/L) and recently active (1.4 nM/L) patient groups were greater than that of the inactive group (0.6 nM/L; p < 0.05). CONCLUSIONS: Evidence of coagulation cascade activation in patients with active and recently active IBD suggests an increased risk of thrombogenesis during active disease that extends for a period of time after commencement of medications to induce remission in their disease process. It may be prudent to counsel patients to avoid risk factors associated with thrombotic phenomenon around the time of active IBD.

Adolescent↗
PubMed1996

Ventricular enlargement associated with linkage marker for schizophrenia-related disorders in one pedigree.

We previously obtained evidence indicating a genetic linkage marker for schizophrenia and related disorders (two-point lod score = 3.72, P = 0.01) on the short arm of chromosome 5(5p14.1-13.1) in one large pedigree. Automated computer algorithms were used to edge the brain and measure the volume of the ventricles, regional sulcal atrophy, and skull size and shape in the original nuclear family members. Of the 11 subjects who underwent computed tomography, six (three schizophrenic, two with schizotypal personality disorder, and one unaffected) carried the marker allele that co-segregated with schizophrenia-related disorders, while five (all unaffected) did not. The family members with the marker allele linked to schizophrenia-related disorders (n = 6) had significantly (P < 0.05) larger ventricle-brain ratios (VBRs) and more fronto-parietal atrophy (controlling for age) than the family members lacking the schizophrenia-related marker allele (n = 5). The three individuals with the largest VBRs all carried the marker, although they received diagnoses of no schizophrenia-related disorder, schizotypal personality disorder, and schizophrenia. Regional cortical values indicative of cerebrospinal fluid content were higher in the frontal and parietal regions of family members carrying the marker. The hypothesis that genetic linkage is associated with structural brain pathology is difficult to test because of all the potential compounding factors. Our findings suggest the possibility that, in this family, relatively enlarged VBR and fronto-parietal atrophy, as determined by computed tomograph, may be associated with a schizophrenia-related gene and present susceptibility to schizophrenia-related disorders. In addition to a replication of these findings in other similarly linked families yet to be identified, further studies using higher resolution structural and functional neuroimaging techniques will be required.

Brain↗
PubMed1995

Tau isoform expression and phosphorylation state during differentiation of cultured neuronal cells.

The axonal microtubule-associated protein, tau, is thought to play an important role in axonal growth and in the establishment of neuronal polarity. In adult human brain there are six alternatively spliced tau isoforms, which have different microtubule binding affinities in vitro. The tubulin-tau interaction is further modified by phosphorylation of tau and, compared to adult brain tau, both foetal brain tau and paired helical filament (PHF) tau, characteristic of Alzheimer's disease, are hyperphosphorylated. In vivo both the expression of tau isoforms and their phosphorylation states are developmentally regulated. In order to establish the correlation between the expression of tau isoforms and their pattern of phosphorylation, we have characterised these two features in several in vitro models of neuronal differentiation, including the human neuroblastoma cell lines, SK-N-SH, SH-SY5Y and IMR32 cells, rat PC12 cells and primary rat cortical neurones. Sensitive RT-PCR analysis revealed a different complement of tau isoforms in the different cell lines and neuritogenesis was associated mainly with an increase in the overall tau protein level with no apparent phosphorylation changes. A switch in tau isoform expression occurred only at the terminal stages of neuronal development, when it may be important in reinforcing the previously established axonal cytoarchitecture.

Alzheimer Disease↗
PubMed1995

Evidence for the key role of the adipocyte cGMP-inhibited cAMP phosphodiesterase in the antilipolytic action of insulin.

Enhancement of cAMP degradation by increased cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) activity is thought to be an important component of the mechanism whereby insulin counteracts catecholamine-induced lipolysis in adipocytes. In this study the selective cGI-PDE inhibitor OPC3911 was used to evaluate this role of cGI-PDE activation in intact rat adipocytes with special reference to changes in cAMP levels measured as cAMP-dependent protein kinase (cAMP-PK) activity ratios. OPC3911 completely blocked (IC50 = 0.3 microM) the maximal inhibitory effect of insulin on noradrenaline-induced lipolysis and the net dephosphorylation of hormone-sensitive lipase and other intracellular target proteins for insulin action, whereas insulin-induced lipogenesis was not changed. The effect of OPC3911 on cAMP-PK activity ratios at different levels of lipolysis achieved by noradrenaline stimulation revealed that the reduction of cAMP-PK caused by 1 nM insulin was completely blocked by 3 microM OPC3911. The effect of OPC3911 was not due to an excessive increase in cellular cAMP resulting in 'supramaximal' lipolysis unresponsive to insulin. These data demonstrate that reduction in cAMP levels by the activation of cGI-PDE may be sufficient to account for the antilipolytic action of insulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Nucleotide sequence determination and genetic analysis of the Bacteroides plasmid, pBI143.

The nucleotide sequence and genetic organization of the Bacteroides plasmid pBI143 were determined. The plasmid was 2747 base pairs (bp) and had a G+C content of 41% (GenBank Accession No. U30316). There were two open reading frames greater than 50 codons and these were designated mobA and repA. A 56-bp inverted repeat divided pBI143 into modules with repA and mobA in separate regions. There was a marked difference in the G+C content and codon usage for the two regions; repA had 33% G+C and mobA was 44% G+C. MobA had homology to other Bacteroides mobilization proteins and RepA shared homology to a replication protein from Zymomonas mobilis plasmid pZM2. These two putative replication proteins formed a subgroup of the rolling-circle replication.proteins belonging to the pSN2 family of gram-positive plasmids. Consistent with this finding, single-stranded pBI143 DNA was detected in plasmid containing Bacteroides fragilis cultures. Availability of the pBI143 sequence allowed the elucidation of the complete nucleotide sequence for pFD288 an 8.9-kb Bacteroides shuttle vector (GenBank Accession No. U30830).

Amino Acid Sequence↗
PubMed1995

High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions.

The 1.4 kb 5' polygalacturonase (PG) gene-flanking region has previously been demonstrated to direct ripening-specific chloramphenicol acetyl transferase (CAT) expression in transgenic tomato plants. The steady state level of CAT mRNA in these plants was estimated to be less than 1% of the endogenous PG mRNA. Further constructs containing larger PG gene-flanking regions were generated and tested for their ability to direct higher levels of reporter gene expression. A 4.8 kb 5'-flanking region greatly increased levels of ripening-specific reporter gene activity, while a 1.8 kb 3' region was only shown to have a positive regulatory role in the presence of the extended 5' region. Transgenic plants containing the CAT gene flanked by both of these regions showed the same temporal pattern of accumulation of CAT and PG mRNA, and steady-state levels of the transgene mRNA were equivalent to 60% of the endogenous PG mRNA on a per gene basis. The proximal 150 bp of the PG promoter gave no detectable CAT activity. However, the distal 3.4 kb of the 4.8 kb 5' PG promoter was shown to confer high levels of ripening-specific gene expression when placed in either orientation upstream of the 150 bp minimal promoter. The DNA sequence of the 3.4 kb region revealed a 400 bp imperfect reverse repeat, and sequences which showed similarity to functionally significant sequences from the ripening-related, ethylene-regulated tomato E8 and E4 gene promoters. The possible roles of the flanking regions in regulating PG gene expression are discussed.

Base Sequence↗
PubMed1995

Dosimetric effects of matching electron fields with cobalt 60 fields in the management of head and neck cancer.

The dose distributions in a blocked Co 60 beam with the area under the block irradiated with a 9-MeV electron beam at a nominal source to surface distance (SSD) of 100 cm and at an extended SSD of 115 cm were determined. The photon block was simulating the spinal cord shielding used when treating the posterior neck. A hot spot of 10% appeared close to the junction on the side of the photon field when electron treatment was given at a nominal SSD of 100 cm. At 115 cm SSD, the magnitude of the hot spot on the side of photon field increased to 20% and a cold spot on the order of 10% appeared in the distal area of the irradiated volume treated with electrons. A gap of 0.5 cm between the light field edges of the photon field and the electron field reduced the hot spot from 20% to 10% on the side of photon field. However, the magnitude of the cold spot on the side of electron field increased to 20%, and it appeared across the irradiated volume. Considering the limitation of the design of the electron applicators, which precludes the use of a nominal SSD of 100 cm for head and neck treatments, it is recommended that no gap should be allowed between the photon fields and the extended SSD electron fields. Clinically a limited hot spot of 20% is considered more acceptable than a cold spot of 20% extending from proximal to distal areas of the treatment volume.

Cobalt Radioisotopes↗
PubMed1995

Immediate-early gene expression in human saphenous veins harvested during coronary artery bypass graft operations.

Saphenous vein graft occlusion is a common late complication of coronary bypass grafting. Intimal smooth muscle cell hyperplasia is a component of this pathobiology, but the underlying molecular events are poorly understood. Immediate-early genes are activated shortly after growth stimulation and subserve cellular functions, which may contribute to intimal smooth muscle cell accumulation. In the present study, human saphenous vein grafts were harvested with minimal manipulation during coronary bypass and processed for isolation of total ribonucleic acid to examine change in immediate-early gene expression of messenger ribonucleic acid by Northern blotting techniques. Thirty saphenous vein grafts were incubated at 4 degrees C in Dulbecco's modified Eagle media from 30 minutes to 10 hours. The messenger ribonucleic acids for immediate-early genes c-fos and c-myc were weak or undetectable in controls but were increased (> 10 times controls) within 1 hour (c-fos) and persisted for at least 6 hours (c-myc) after harvest. Our results demonstrate, for the first time in human vascular tissue, incipient immediate-early gene induction. This information may lead to molecular therapies to control saphenous vein graft disease.

Adult↗
PubMed1995

Axillary sampling and radiotherapy does not compromise the patient with early breast cancer: Trafford database experience.

Local recurrence has been studied in a selected group of 379 patients, 39% of whom underwent simple mastectomy and 61% lumpectomy. The axilla was sampled in 52%, cleared in 8% and untreated in 40%. Axillary recurrence occurred in 16% of patients with a median follow-up of 54 months. Ninety-one per cent of these patients with lymph node recurrence had no surgery or sampling to the axilla, and a third received radiotherapy. Local recurrence was satisfactorily controlled by various modalities of treatment in all but 29 patients. Death related to distant metastasis in all but six of these 29 patients. Analysis suggests that patients with locally uncontrolled disease had aggressive tumours at diagnosis which were larger than average, most often grade 3 histologically and more frequently required mastectomy. Our results support previous studies indicating that survival would not be influenced by more radical surgery but that sampling followed by radiotherapy would have prevented the majority of our lymph node recurrences.

Adult↗
PubMed1995

Intestinal mucin inhibits adhesion of human enteropathogenic Escherichia coli to HEp-2 cells.

Mucins are complex glycoproteins that cover the intestinal mucosal surface and are thought to provide protection against enteropathogenic infections by preventing bacterial adherence and subsequent colonization, invasion and toxin delivery. The aims of this study was to evaluate the capacity of intestinal mucin to inhibit epithelial cell adhesion by human enteropathogenic Escherichia colI (EPEC). EPEC strain E2348/69 (serotype O127:H6), known to produce a 94-kd epithelia cell-adhesion-mediating outer membrane protein called intimin and known to contain an EPEC adherence factor (EAF) plasmid, and three of its mutants were studied. Crude mucus was obtained from rabbit small-ubtestinal mucosal scrapings. Purified mucins were isolated by serial cesium chloride density gradient ultracentrifugation. Bacterial adhesion to HEp-2 epithelial cell monolayers in the presence of purified intestinal mucins was quantified by recovery of live viable bacteria as colony-forming units. EPEC strain E2348/69 adhesion was inhibited in a concentration-dependent manner by increasing amounts of mucin (adhesion suppressed to <1% by 150 microgram mucin). The relative magnitudes of inhibition of adhesion of E2348/69 and its mutants [JPN15 (EAF plasmid negative), JPN15.96(pMAR7) (intimin negative), and JPN15.96 (intimin negative, EAF plasmid negative)] were similar in the presence of equal amounts of mucin. The presence of the intimin protein and EAF plasmid are not necessary to the proficiency of mucin to inhibit HEp-2 epithelial cell adhesion.

Animals↗
PubMed1995

Heat shock protein studies in type 1 and type 2 diabetes and human islet cell culture.

Heat shock proteins (HSP) play an important role in auto-immunity and infection. Glutamic acid decarboxylase (GAD) the prime antigen in Type 1 diabetes has similar amino acid sequences to HSP65. An ELISA was developed using a plant-derived HSP65 antibody. HSP65 antibody was present in the serum of all normal subjects (median 1.64 AU, IQ range 1.49-1.74). Lower levels were found in established Type 1 diabetes (1.41 AU, 1.32-1.61, p < 0.001) and Type 2 diabetes (1.45 AU, 1.35-1.59, p < 0.006). In Type 1 HSP antibody levels fell with age (p = 0.007) and with duration (p = 0.008) and women with Type 1 had lower levels than men (p = 0.009). Human islet cell culture subjected to heat shock revealed an approximate four fold increase in heat shock protein antigen in the surrounding medium. The release of HSP antigen from stressed islet cells together with the finding of HSP antibody in the serum of all subjects suggest that HSP65 should not be completely discarded as having a possible role in the development of Type 1 diabetes. Low levels of HSP antibody in patients with established diabetes is probably a manifestation of impaired immunity induced by the diabetic state.

Adolescent↗
PubMed1995

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.

Amino Acid Sequence↗