Biomedical subjects

A M Cook

Publications and source records attributed to A M Cook.

At least 55 records · Page 3Linked to original sources

PubMed1994

Microbial metabolism of sulfur- and phosphorus-containing xenobiotics.

The enzymes involved in the microbial metabolism of many important phosphorus- or sulfur-containing xenobiotics, including organophosphate insecticides and precursors to organosulfate and organosulfonate detergents and dyestuffs have been characterized. In several instances their genes have been cloned and analysed. For phosphonate xenobiotics, the enzyme system responsible for the cleavage of the carbon-phosphorus bond has not yet been observed in vitro, though much is understood on a genetic level about phosphonate degradation. Phosphonate metabolism is regulated as part of the Pho regulon, under phosphate starvation control. For organophosphorothionate pesticides the situation is not so clear, and the mode of regulation appears to depend on whether the compounds are utilized to provide phosphorus, carbon or sulfur for cell growth. The same is true for organosulfonate metabolism, where different (and differently regulated) enzymatic pathways are involved in the utilization of sulfonates as carbon and as sulfur sources, respectively. Observations at the protein level in a number of bacteria suggest that a regulatory system is present which responds to sulfate limitation and controls the synthesis of proteins involved in providing sulfur to the cell and which may reveal analogies between the regulation of phosphorus and sulfur metabolism.

Bacteria↗

PubMed1994

Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

Comamonas testosteroni T-2, grown in terephthalate (TER)-salts medium, synthesizes inducible enzymes that convert TER to (1R,2S)-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD) and protocatechuate (PC). Anion-exchange chromatography of cell extracts yielded two sets of fractions, R and Z, that were necessary for oxygenation of TER to DCD; we termed this activity the TER dioxygenase system (TERDOS). An NAD(+)-dependent DCD dehydrogenase, which converted DCD to PC, overlapped all fractions R. No significant purification from fraction R, which contained an NADH-dependent reductase function(s) of TERDOS, was attained. Fraction Z, at the end of the gradient, contained essentially one protein, which was further purified by hydrophobic interaction chromatography. This component, Z, had the UV-visible spectrum and electron paramagnetic resonance characteristics of a Rieske [2Fe-2S] protein and was considered to be the oxygenase. M(r)s of about 126,000 for oxygenase Z under native conditions were observed. Oxygenase Z consisted of two subunits, alpha and beta, with M(r)s of 49,000 and 18,000, respectively, under denaturing conditions. We presume that this oxygenase has an alpha 2 beta 2 structure. The sequences of the N-terminal amino acids of each subunit were determined. The activity of the purified enzyme was enhanced about fivefold by addition of Fe2+. In the presence of O2, NADH, and fraction R, component Z catalyzed the stoichiometric transformation of TER to PC, with the intermediate formation of DCD. The reaction was confirmed as a dioxygenation when we observed incorporation of two oxygen atoms from 18O2 into PC. The substrate range of TERDOS appeared to be narrow; apart from TER, only 2,5-dicarboxypyridine and 1,4-dicarboxynaphthalene (of 11 compounds tested) were converted to a product.

Biodegradation, Environmental↗

PubMed1994

Reproducibility of bone ages when performed by radiology registrars: an audit of Tanner and Whitehouse II versus Greulich and Pyle methods.

This audit analysed the Tanner and Whitehouse II twenty bone (TW2) method of bone age assessment which was used in our department, and compared it with the Greulich and Pyle (GP) method. 50 previous bone ages were independently re-calculated by each of three registrars using both techniques, with the time taken to perform each assessment being recorded. For each method the interobserver variation was analysed in terms of the spread of results. The intraobserver variation in TW2 was determined by comparing the bone age originally reported with that subsequently calculated on the same film by the same registrar. The average spread of results was 0.74 years for TW2 method, and 0.96 years for the GP method and this difference is not statistically significant at the 5% level. The average intraobserver variation to TW2 was 0.33 years, but with 95% confidence limits of -0.87 to +1.53 years. The average time taken was 7.9 min for TW2 and 1.4 min for GP assessments. It was concluded that the GP method gave similar reproducibility and was faster than the TW2 method. Following clinical discussion the routine departmental bone age assessment method was changed from the TW2 to the GP method.

Age Determination by Skeleton↗

PubMed1994

Dynamic contrast-enhanced and fat suppressed magnetic resonance imaging in suspected recurrent carcinoma of the breast: preliminary experience.

20 women with suspected recurrent breast cancer who had undergone previous breast-conserving operations were investigated using dynamic contrast-enhanced gradient echo (GRE) and fat suppressed spin echo (SE) magnetic resonance (MR) imaging. Histologically confirmed recurrent tumour was readily recognized on dynamic GRE scans by virtue of rapid, early and avid enhancement. Benign scars enhanced more slowly and reached lower magnitudes of enhancement. Fat suppressed SE images, which were typically acquired 10 min after contrast administration, were sensitive for the detection of tumour recurrence but lacked specificity. Early scanning after contrast administration offers the best prospects for distinguishing tumour recurrence from benign scarring. The criteria used to distinguish these two entities are highly dependent on the scan technique and the time at which images are obtained post-contrast.

Aged↗

PubMed1993

Purification of two isofunctional hydrolases (EC 3.7.1.8) in the degradative pathway for dibenzofuran in Sphingomonas sp. strain RW1.

Sphingomonas sp. strain RW1, when grown in salicylate-salts medium, synthesized the enzymes for the degradation of dibenzofuran. The reaction subsequent to meta cleavage of the first benzene ring was found to be catalyzed by two isofunctional hydrolases, H1 and H2, which were purified by chromatography on anion exchange, hydrophobic interaction and gel filtration media. Each enzyme was able to hydrolyze 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)hexa-2,4-dienoate and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate to produce salicylate and benzoate, respectively. SDS/PAGE of each purified enzyme showed a single band of M(r) 31,000 (H1) or 29,000 (H2). The N-terminal amino acid sequences of the two proteins showed 50% homology.

Bacteroidetes↗

PubMed1993

The use of the Wallstent in aorto-iliac vascular disease.

Forty-three patients had self-expanding endovascular Wallstents inserted for aorto-iliac arterial disease. A total of 59 Wallstents were inserted into 50 iliac arteries. There were 27 males and 16 females with a mean age of 62.51 years. Included in the series were 19 iliac occlusions, 21 severe atheromatous disease of the aortoiliac segment, two severe dissections following conventional angioplasty and one re-stenosis of a previously dilated iliac segment. All of the stents were successfully deployed but there were five significant complications. One case of distal embolism was treated by surgical embolectomy but the other four were treated by an additional percutaneous technique. At follow-up after 6 months all patients remain symptomatically improved. Aorto-iliac stenting is a cost-effective treatment in selected cases of severe aorto-iliac disease.

Aortic Diseases↗

PubMed1993

The use of the Wallstent endovascular prosthesis in the treatment of malignant obstruction of the superior vena cava.

Seventeen patients (10 men and seven women) aged 48-76 years (mean 63.4) who were suffering from malignant superior vena caval obstruction had Wallstents implanted percutaneously to relieve the obstruction. Fifteen of the patients had previously been treated by radiotherapy or chemotherapy, with either failure to remit or later recurrence. Six patients required thrombolysis prior to stenting. All patients had rapid relief of the obstructive symptoms. This was sustained in 15 patients to date or at death. Two patients, both of whom had pre-stent lysis, developed stent thrombosis following withdrawal of anticoagulation at 4 months and 7 days respectively. Stenting has been shown to be extremely effective at relieving superior vena caval obstruction in these patients and is usually the only method available in this condition when other treatments fail. Whether all patients presenting with malignant superior vena caval obstruction should be stented de novo remains to be proven.

Aged↗

PubMed1993

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.

Bacteria, Anaerobic↗

PubMed1993

Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system.

Sphingomonas sp. strain RW1 synthesized a constitutive enzyme system that oxygenated dibenzofuran (DBF) to 2,2',3-trihydroxybiphenyl (THB). We purified this dibenzofuran 4,4a-dioxygenase system (DBFDOS) and found it to consist of four components which catalyzed three activities. Two isofunctional, monomeric flavoproteins (components A1 and A2; M(r) of about 44,000) transferred electrons from NADH to the second component (B; M(r) of about 12,000), a ferredoxin, which transported electrons to the heteromultimeric (alpha 2 beta 2) oxygenase component (C; M(r) of alpha, 45,000; M(r) of beta, 23,000). DBFDOS consumed 1 mol each of NADH, O2, and DBF, which was dioxygenated to about 1 mol of THB; no intermediate was observed. The reaction was thus the dioxygenation of DBF at the 4 and 4a positions to give a diene-diol-hemiacetal which rearomatized by spontaneous loss of a phenolate group to form THB. Components A1 and A2 each reduced dichlorophenolindophenol but had negligible activity with cytochrome c; each lost the yellow color, observed to be flavin adenine dinucleotide, upon purification. Component B, which transported electrons to the oxygenase or cytochrome c, had an N-terminal amino acid sequence with high homology to the putidaredoxin of cytochrome P-450cam. The oxygenase had the UV spectrum of a Rieske iron-sulfur center. We presume DBFDOS to be a class IIA dioxygenase system (EC 1.14.12.-), functionally similar to pyrazon dioxygenase.

Amino Acid Sequence↗

PubMed1993

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2.

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.

Aerobiosis↗

PubMed1993

Proteins induced by sulfate limitation in Escherichia coli, Pseudomonas putida, or Staphylococcus aureus.

Two-dimensional gel electrophoresis of proteins from Escherichia coli, Pseudomonas putida, and Staphylococcus aureus, grown with methionine or one of a variety of organosulfates and organosulfonates as the sole source of sulfur, showed expression of specific sets of 7 to 14 proteins which were not observed during growth with sulfate or cysteine for all three species or with thiocyanate for P. putida and S. aureus. Under the same conditions, arylsulfatase activity in P. putida and S. aureus was seen to increase by up to 140-fold, suggesting that the proteins induced under these conditions may be involved in sulfur metabolism. We propose that these proteins are members of a sulfate starvation-induced stimulon.

Arylsulfatases↗

PubMed1993

A study of the application of Fitts' law to selected cerebral palsied adults.

Fitts' Law was applied to the evaluation of discrete aimed arm movements in one dimension performed by 6 adults with cerebral palsy and 6 adults with normal movement. Targets (1.27, 3.81, 6.35, 8.89 cm in width) placed at distances of 20.32, 30.48, 40.64, and 50.80 cm provided Indices of Difficulty of 2.19 to 6.32 bits. A video game was used for the signal to move. The t tests for corrected means showed that the cerebral palsied group had greater reaction and movement times than than the normal group. Linear regression analyses for pooled data and for the individual cerebral palsied subjects showed (a) no relationship between reaction time and Index of Difficulty for either group and (b) a negative, linear relationship between reaction time and Index of Difficulty for 1 cerebral palsied subject. Movement time was linearly and positively related to the Index of Difficulty for the normal group but not for the cerebral palsied group. Two cerebral palsied subjects had movement times which conformed to Fitts' Law. While the exceptions to Fitts' Law may be due to the limited range of movement and spasticity caused by severe cerebral palsy, there are indications that two of the cerebral palsied subjects were performing ballistic rather than visually guided aimed movements. Fitts' Law may still apply when the distances and target positions are individualized to conform to subjects' specific limitations and when the signal to move is auditory rather than visual.

Adult↗

PubMed1992

Aeromonas spp as a potential cause of diarrhoea in children.

AIMS: To determine the prevalence of Aeromonas spp in the faeces of children and the association with symptoms of gastroenteritis. METHODS: Faecal specimens (n = 1026) were cultured for Aeromonas spp using three selective media and an enrichment broth at both 30 degrees C and 37 degrees C. The isolation of Aeromonas spp was correlated with symptoms of gastroenteritis, previous antibiotic use, and environmental temperature. RESULTS: Aeromonas spp (n = 28) from 26 (2.5%) patients were recovered. Bile salt, Irgasan, and brilliant green agar was the most efficient selective culture medium. Eleven of the patients had symptoms of gastroenteritis, usually mild diarrhoea of two to three days' duration, in the absence of other recognised enteropathogens. A caviae was a particularly frequent isolate (nine out of 11 cases) in symptomatic individuals. Only one out of seven Aeromonas spp recovered by enrichment culture alone was possibly associated with symptoms of gastroenteritis. There was a close correlation between the environmental temperature and isolation of Aeromonas spp. CONCLUSIONS: Aeromonas spp and particularly A caviae may cause gastroenteritis in children, most often during warmer months of the year. Culture for these potential enteropathogens could be confined to summer and autumn months. Bile salt, Irgasan, and brilliant green selective agar, but not alkaline peptone water enrichment, is an efficient culture medium for recovering possibly clinically important isolates.

Aeromonas↗

PubMed1992

Phenotypic methods for speciating clinical Aeromonas isolates.

AIMS: To establish the suitability of currently available phenotypic methods for speciation of clinical Aeromonas isolates in diagnostic microbiology laboratories. METHODS: Using 62 Aeromonas spp, three schemes based on biochemical reactions were compared: a series of conventional tests; a system based on the suicide phenomenon, comprising two tubes in total; and a commercially available test, API 20 NE, augmented with a plate assay for beta haemolysin production. The whole cell and outer membrane protein (OMP) profiles of strains were examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE), according to the results of the above schemes, to determine the intra-species homogeneity. RESULTS: Ninety per cent of strains were identified satisfactorily according to conventional criteria. For these strains, agreement was obtained using the suicide phenomenon and API schemes in 93% and 88% of cases, respectively. The three schemes concurred for 82% of strains. Whole cell protein profiles were unsuitable for comparing strains within a species. However, OMP patterns were similar for 89% of A caviae and 63% of A hydrophila. CONCLUSION: Phenospeciation of clinical Aeromonas isolates by the scheme based on the suicide phenomenon is simple to perform and accurate, and suitable for use in the diagnostic laboratory. OMP profiles are potentially useful for confirming the identity of A caviae and most A hydrophila, but not A sobria.

Aeromonas↗

PubMed1992

Audit in action: significant reduction of double-contrast barium enema screening time with no loss of examination quality.

The double-contrast barium enema is an examination of high radiation exposure to the patient. As part of a departmental audit, our own fluoroscopic screening times were being recorded. A study was undertaken to see if a determined effort could reduce the screening time without having a detrimental effect on the quality of the examinations. Methods of screening time reduction were identified and then implemented by two radiologists. In the period before the study the average screening time for radiologists A and B was 2.6 min and 3.2 min, respectively (over 56 examinations). After implementation of the changes, the average screening time for both was 0.9 min (over 50 examinations). This is a statistically significant reduction (p less than 0.01). The examinations were assessed independently by a gastroradiologist. No difference in the quality of the examinations was identified. We conclude that a significant reduction of screening time for the double-contrast barium enema can be achieved without loss of examination quality.

Barium Sulfate↗

PubMed1991

4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2.

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

Amino Acids↗

PubMed1991

Dichloromethane utilized by an anaerobic mixed culture: acetogenesis and methanogenesis.

Dichloromethane (8.9 mg/l) was eliminated from industrially polluted, anaerobic groundwater in a fixed-bed reactor (43 m3) which was packed with activated charcoal and operated continuously for over three years. The elimination of dichloromethane over this period was some ten-fold in excess of the sorptive capacity of the charcoal, and the elimination (3.7 mg/h.[kg of charcoal]: residence time, 49 h) was tentatively attributed to dehalogenative microorganisms immobilized on the charcoal. Anaerobic enrichment cultures, with dichloromethane as the sole added source of carbon and energy, were inoculated with material from the reactor. Reproducibly complete substrate disappearance in subcultures was observed when traces of groundwater (1%) or yeast extract (0.01%) were supplied. Fed-batch experiments under an atmosphere of CO2 plus N2 led to the conversion in 11 days of 11 mM dichloromethane to 3 mM acetate and 2 mM methane, with a growth yield of 0.4 g of protein/mol of dichloromethane; insignificant amounts (less than 1 microM) of chloromethane accumulated. Methanogenesis could be inhibited by 50 mM 2-bromoethane sulfonate without any effect on the dehalogenation rate. The maximum dehalogenation rate was 0.13 mmol dichloromethane/h.l (2.6 mkat/kg of protein).

Acetates↗

PubMed1991

4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfontate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. Mrs of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from 18O2 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde.

Amino Acids↗