Search PubMed⌕ Search

Biomedical subjects

C M King

Publications and source records attributed to C M King.

At least 55 records · Page 3Linked to original sources
PubMed1996

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids.

Genes for the 290 amino acid, 33-34 kDa cytosolic acetyltransferases (NAT1* and NAT2*) from rat and hamster were cloned and expressed in Escherichia coli. Active clones were selected by a simple visual test for their ability to decolorize 4-aminoazobenzene in bacterial medium by acetylation. These recombinant acetyltransferases were analyzed for: (i) N-acetyltransferase, which was assayed by the rate of acetyl coenzyme A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N,O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene. Both NAT2s showed first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 microM, with apparent K(m) values of 22-32 and 62-138 microM respectively. Although under the same conditions the N-acetylation rates for the two NAT1s declined by > 50%, below 5 microM 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, and the apparent K(m) values were 0.2-0.9 microM. For N,O-acyltransferase, the apparent K(m) values of the NAT1s were approximately 6 microM, while the K(m) values of the NAT2s were approximately 20- to 70-fold higher. SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MWr) of approximately 31 kDa for both NAT1s and rat NAT2 and approximately 29 kDa for hamster NAT2. Comparable MWr values were observed for native hamster liver NAT1 and NAT2 and for rat NAT1 under the same conditions. Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data show that the rat NAT2* gene does code for a functional acetyltransferase, with properties similar to those of hamster liver NAT2. The data also indicate that at low substrate concentrations, NAT1 would apparently play the predominant role in vivo in N-acetylation and N,O-acyltransfer of aromatic amine derivatives, including their metabolic activation to DNA-reactive agents.

Acetylation↗
PubMed1995

An investigation of antioxidant status, DNA repair capacity and mutation as a function of age in humans.

We are constantly exposed, throughout life, to a wide variety of extrinsic and intrinsic agents which have the potential to damage cellular biomolecules, including DNA. Imperfections in cellular defence systems which protect against the fixation of DNA damage can lead to an accumulation of mutations which on their own, or in combination with other age-related changes, may contribute to ageing and the development of age-related pathologies. We have previously reported an increase in frequency of mutation with age in human lymphocytes taken from healthy males in the age groups, 35-39, 50-54 and 65-69 years. In this article we report on the findings of a recent study which was designed to assess whether the age-related increase in frequency of mutation was due to a decreased efficacy of the defence systems against ROS-induced DNA damage, namely antioxidant status and DNA repair processes, in the same study subjects. In vivo antioxidant status was assessed in each of the study subjects by measuring blood levels of; superoxide dismutase (SOD; EC 1.15.1.1), glutathione peroxidase (GPx; EC 1.11.1.9), catalase (EC 1.11.1.6), caeruloplasmin (CPL), uric acid and bilirubin. We did not find any statistically significant differences in the mean levels of these antioxidants between the three different age groups. To investigate the efficacy of DNA repair processes against ROS-induced DNA damage, an ELISA was used to quantitate DNA damage (as % single-stranded DNA; %SS-DNA) at various times following treatment of peripheral blood lymphocytes with hydrogen peroxide (H2O2). The results of this part of the study showed that in untreated lymphocytes, basal levels of %SS-DNA were significantly higher in individuals from the 65-69 years age group compared to the 35-39 years age group (p = 0.039, 0.0013; at 5% level of significance). No significant differences were found in H2O2 susceptibility with age immediately following treatment (p = 0.71, 1.00; at 5% level of significance) but a consistent and significant increase was observed in %SS-DNA remaining 90 min post-treatment in lymphocytes from subjects in the 65-69 years age group, compared to %SS-DNA present in lymphocytes from the 35-39 years age group (p = 0.013, 0.024; at 5% level of significance). The results of this study suggest that the age-related increase in frequency of mutations is not contributed to by alterations of in vivo antioxidant status with age but is by a decreased efficacy of the repair of ROS-induced DNA damage with age. The biological implications of somatic mutations in the ageing process are discussed.

Adult↗
PubMed1995

The significance of ipsilateral leg ischaemia after renal transplantation.

We present three renal transplant patients who have been investigated for leg ischaemia on the side of the transplant. All were men aged between 50 and 57 years. Two had an iliac stenosis proximal to the transplant kidney and were treated successfully by percutaneous transluminal angioplasty. The other, with an internal iliac anastomosis, had occlusion of the external iliac artery and underwent femoro-femoral bypass grafting. Eight years later, almost 17 years after transplantation, this hyperlipidaemic patient was found to have an internal iliac origin stenosis proximal to the transplant kidney and also underwent successful angioplasty. In patients with functioning renal transplants, symptoms of arterial insufficiency in the ipsilateral leg should be investigated urgently because a proximal iliac stenosis potentially threatens graft survival.

Arterial Occlusive Diseases↗
PubMed1995

Carcinogenicity of nitropyrenes in the newborn female rat.

The carcinogenicities of 1-nitropyrene (1-NP), 4-nitropyrene (4-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP), 3-hydroxy-1-nitropyrene (3-OH-1-NP) and a mixture of 6- and 8-hydroxy-1-nitropyrene (6/8-OH-1-NP) were investigated in newborn female rats. Newborn female CD rats were treated s.c. eight times at weekly intervals with a total dose of 6.3 mumol 1-NP,1,3-DNP,1,6-DNP or 1,8-DNP; control animals received only dimethylsulfoxide (DMSO). The experiment was terminated at 67 weeks. With the exception of 1,6-DNP- and 1,8-DNP-treated animals, which had average survival periods of 149 and 164 days respectively, the animals administered the other compounds did not show decreased survival. Malignant fibrous histiocytomas were observed in 12%, 100% and 100% of the rats treated with 1,3-, 1,6- and 1,8-DNP respectively. Leukemia was found in 20% and 22% of the animals treated with 1,6- and 1,8-DNP respectively. No control rats developed these tumors. Additionally, mammary tumors were induced in rats treated with 1-NP. Newborn female CD rats were similarly treated with 1-NP, 4-NP, 3-OH-1-NP, 6/8-OH-1-NP or DMSO and newborn female F344 rats were treated with 1-NP or DMSO. The experiment was terminated at 86 weeks, 1-NP and 4-NP produced mammary adenocarcinoma in CD rats. Although 1-NP did not produce mammary adenocarcinoma in F344 rats, it induced leukemia. 4-NP also induced malignant fibrous histiocytomas in CD rats. This study demonstrates that 4-NP is more carcinogenic than 1-NP and that CD rats are more susceptible than F344 rats to mammary carcinogenesis by 1-NP. Additionally, 1,6- and 1,8-DNP are more potent than 1-NP in inducing malignant fibrous histiocytomas and leukemia.

Animals↗
PubMed1995

Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1.

Sequence analyses have revealed the existence of homology between certain aeroallergens and proteolytic enzymes. This homology can be expressed functionally, but its significance to airway pathophysiology is unknown. Studies with Madin-Darby canine kidney cells and canine tracheal epithelial cells grown on plastic substrata or matrix proteins suggest that Der p1, a major allergen from the house dust mite Dermatophagoides pteronyssinus and a cysteine proteinase, or the unfractionated growth medium extract (SGME) from which it was purified, are both capable of causing cell detachment. The ability of both agents to produce functional changes in the barrier function of the epithelium was further demonstrated using isolated bovine airway preparations. Over a 3-h duration, both Der p1 and SGME elicited significant increases in the permeability of isolated sheets of bronchial mucosa to serum albumin. Exposure of isolated bronchial segments to luminally applied solutions of Der p1 resulted in histologic evidence of epithelial injury. Neither Der p1 nor SGME was active in these experimental systems unless chemically reduced, suggesting that the effect was initiated by cysteine proteinase activity. Similar augmentation of mucosal permeability and tissue injury was produced by bovine trypsin and collagenase from Clostridium histolyticum. In both the isolated mucosal sheet model and in cultured cells, the actions of Der p1 or SGME were associated with relatively little cytolysis, suggesting a specific action of the reagents on cell attachment. These results demonstrate a new functional activity of Der p1 that may be germane to the processes of allergen presentation, inflammatory cell activation, and chronic tissue injury.

Animals↗
PubMed1995

Induction of 70-kD heat shock protein in scleractinian corals by elevated temperature: significance for coral bleaching.

In this study, the induction of the 70-kD family of heat shock proteins (hsp70) has been examined in stony coral tissues. In these experiments, the only difference from control conditions has been exposure to a temperature approximating that at which field bleaching in the Caribbean is known to occur, approximately 30 degrees C or 1 degree-2 degrees C above long-term average seasonal maximum temperatures. A constitutive hsp70 has been identified both in the zooxanthellate (hermatypic) coral, Montastrea annularis, and in two corals lacking symbiotic algae, Tubastrea cocchinea and Astrangia danae (Cnidaria, Anthozoa, Scleractinia). Western blots of experimental tissues fractionated by polyacrylamide gel electrophoresis indicate that the initial induction of hsp70 occurs rapidly, within one hour of transfer to water of elevated temperature. Thereafter, the level of hsp70 decreases within 12-24 hours to approximately the constitutive level. In field-bleached specimens of M. annularis, hsp70 is not detected. Since this coral tissue, once bleached to whiteness, contains no 70-kD heat shock protein, we conclude that the process of coral bleaching might include, among other metabolic alterations, a failed heat shock response. In addition to being compromised in other normal functions, the bleached coral would lose the capacity to protect itself against environmental stress. The eventual loss of algae by bleached coral is likely to be consequent to several metabolic changes in the coral tissue. However, the uncoupling of that symbiotic relation is not concomitant with the initial stress response of heat shock protein synthesis.

Animals↗
PubMed1994

An investigation of mutation as a function of age in humans.

An accumulation of mutations on their own or together with other age-related changes may contribute to aging and the development of age-related pathologies. The aim of this investigation was to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35-39, 50-54 and 65-69 years). Results show that the mean MF in the 65-69 years group was approximately twice that in the 35-39 and 50-54 years groups (4.1/10(6) cells, 1.9/10(6) cells and 1.79/10(6) cells, respectively) increasing by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65-69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Adult↗
PubMed1994

Imaging islet cell tumours.

The localization of islet cell tumours presents a challenge to the radiologist and requires meticulous attention to detail in both technique and interpretation. As several imaging techniques are capable of demonstrating the tumour and none is absolutely accurate, a rational approach to the localization of these tumours requires a careful consideration of cost, sensitivity and the availability of special expertise. In almost all cases, initial imaging is performed with a combination of transabdominal ultrasound and CT. This will demonstrate the tumour and any hepatic metastases in about 40% of gastrinomas, 80% of insulinomas and almost all other functioning and non-functioning tumours. Where these tests are negative or equivocal, arteriography (which may be combined with ASVS) is the next line of investigation. If the tumour remains undetected, it is likely to be a small insulinoma or gastrinoma. Further investigation is dependent on local practice and the tumour type. Endoscopic ultrasound is rapidly emerging as a technique of high sensitivity in detecting small pancreatic tumours and may also demonstrate extrapancreatic gastrinomas. Transhepatic venous sampling and somatostatin receptor imaging have the advantage that they are not directly dependent on tumour size and they are particularly applicable to difficult cases where other imaging modalities are negative. TPVS is invasive and, while sensitive for insulinomas, is frequently unhelpful in gastrinomas. Somatostatin receptor scintigraphy, on the other hand, is more sensitive for gastrinomas. In future, MRI may prove to be at least as accurate as CT but as yet its exact role is uncertain. At the time of surgery, intraoperative ultrasound is a useful adjunct to palpation, and may avoid a standard distal pancreatectomy in patients with insulinoma.

Adenoma, Islet Cell↗
PubMed1994

Characteristics of a purified dog hepatic microsomal N,O-acyltransferase.

Dog liver microsomes have at least three different enzymes that are capable of the deacylation of amides, N-arylhydroxamic acids and carboxylesters, the acyltransfer of N-arylhydroxamic acids and the N-acetylation of arylamines. As judged by SDS-PAGE stained with silver nitrate, one of these enzymes was purified to homogeneity by sequential treatment with Triton X-100, ion-exchange column chromatography, gel filtration and chromatofocusing. The protein was a glycoprotein trimer with a subunit weight of approximately 60 kDa. It showed microheterogeneity on analytical isoelectric focusing (IEF) in polyacrylamide with pls of 5.4-5.6. Following digestion with endoglycosidase H, its subunit weight was reduced to approximately 58 kDa, and it appeared to be homogeneous on IEF with a pl of approximately 5.6. A monoclonal antibody prepared against this enzyme also reacted with the pl 6.0 carboxylesterase of rat liver microsomes, but did not react with the other two dog hepatic acyltransferases. Conversely, a polyclonal antibody raised against the rat esterase reacted with the dog enzyme. The N-terminal sequence of the enzyme was Y-P-S-L-P-P-V-V-D-T-V-Q-G-K-V-, which was homologous to the form 1 carboxylesterase of rabbit liver and the pl 6.0 carboxylesterase of rat liver. Immunohistochemical analyses showed the presence of this enzyme in the epithelium of dog liver and urinary bladder, human liver and rat liver, esophagus, forestomach, glandular stomach, small and large intestines, renal tubules, trachea and prostate and alveolar cells of lung. Since this enzyme is present in the urothelium, it may be important for the activation of urinary metabolites of carcinogenic arylamines for the initiation of bladder carcinogenesis in the dog.

Acyltransferases↗
PubMed1994

Biochemical and genetic analysis of two acetyltransferases from hamster tissues that can metabolize aromatic amine derivatives.

Cytosolic activities in liver, stomach, small intestine, colon, lung, kidney, brain and spleen of hamster have been shown to be capable of N-acetylation, O-acetylation and N,O-acetyltransfer utilizing aromatic amine derivatives as substrates. These activities, which have been implicated in the metabolic activation and carcinogenicity of these compounds, were highest in the liver and small intestine. N-Acetylation could be demonstrated with either acetyl CoA or N-hydroxy-N-acetylaminoarenes serving as acetyl donors. Each of these tissues gave two immunoreactive bands (approximately 29 and 31 kDa) on Western blots using anti-rat AT monoclonal antibodies for detection. Single copies of two distinct acetyltransferase genes, designated AT1 and AT2, were detected in hamster DNA by Southern blot analysis using gene-specific hybridization probes for the 3' end of the AT coding regions. A third gene with > 80% sequence similarity to codons 118-158 of AT2 was also detected. Sequence analysis of the two AT genes showed that both had intronless, 0.87 kb coding regions. One was identical with the AT1 reported by Abu-Zeid et al. (Abu-Zeid,M., Nagata, K., Miyata,M., Ozawa,S., Fukuhara,M. and Yamazoe,Y., 1991, An arylamine acetyltransferase (AT-1) from Syrian golden hamster liver: cloning, complete nucleotide sequence, and expression in mammalian cells. Mol. Carcinogen., 4, 81-88). The second, AT2 (GenBank accession number L24912), coded for a 290 amino acid sequence that was 79% homologous with hamster AT1 and had a calculated molecular weight of 33.8 kDa, a theoretical pI of 5.96 and the three cysteines that have been conserved in all known vertebrate ATs at positions 44, 68 and 223. Northern blot hybridization using gene-specific AT probes detected mRNAs for both AT1 and AT2 in each of the eight tissues analyzed. Two AT1 transcripts, approximately 1.7 and 2.3 kb, were found in approximately equal ratios in all eight tissues. Three transcripts for AT2, approximately 1.9, 2.1 and 2.4 kb, were present in approximately equal ratios in the brain, small intestine, lung and colon. The liver, stomach, kidney and spleen had primarily the smaller two AT2 mRNAs. The overall abundance of AT mRNAs was highest in liver, small intestine and colon. The coding sequences of all AT mRNAs analyzed were identical to their corresponding genes, although the lengths of both the 5' and 3' untranslated transcript regions varied.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed1994

A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae.

Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.

Allergens↗