Stephen Jeffery

Interleukin-1 Receptor Antagonist Gene Polymorphism and Coronary Artery Disease

BACKGROUND Cytokine gene variations are contributory factors in inflammatory pathology. Allele frequencies of interleukin (IL)-1 cluster genes [IL-1A(-889), IL-1B(-511), IL-1B(+3953), IL-1RN Intron 2 VNTR] and tissue necrosis factor (TNF)-alpha gene [TNFA(-308)] were measured in healthy blood donors (healthy control subjects), patients with angiographically normal coronary arteries (patient control subjects), single-vessel coronary disease (SVD), and those with multivessel coronary disease (MVD). METHODS AND RESULTS Five hundred fifty-six patients attending for coronary angiography in Sheffield were studied: 130 patient control subjects, 98 SVD, and 328 MVD. Significant associations were tested in an independent population (London) of 350: 57 SVD, 191 MVD, and 102 control subjects. IL-1RN*2 frequency in Sheffield patient control subjects was the same as in 827 healthy control subjects. IL-1RN*2 was significantly overrepresented in Sheffield SVD patients (34% vs 23% in patient control subjects); IL-1RN*2 homozygotes in the SVD population (chi2 carriage=8.490, 1 df, P=0.0036). This effect was present though not quite significant in the London population (P=0. 0603). A summary trend test of the IL-1RN SVD genotype data for Sheffield and London showed a significant association with *2 (P=0. 0024). No significant effect of genotype at IL-1RN was observed in the Sheffield or London MVD populations. Genotype distribution analysis comparing the SVD and MVD populations at IL-1RN showed a highly significant trend (P=0.0007) with the use of pooled data. No significant associations were seen for the other polymorphisms. CONCLUSIONS IL-1RN*2 was significantly associated with SVD. A difference in genetic association between SVD and MVD was also apparent.

Characterization of human carbonic anhydrase III from skeletal muscle.

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to β-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.

Lack of c‐kit mutation in familial urticaria pigmentosa

Somatic mutations within c‐kit have been reported in individuals with mastocytoses, including urticaria pigmentosa (UP). We have identified three siblings with UP. We aimed to determine whether the c‐kit proto‐oncogene was playing a part in the aetiology of UP in these three siblings. Using seven microsatellite repeat markers spanning an 8‐cM interval encompassing the c‐kit gene we followed the transmission of the c‐kit gene in this family. Furthermore, single‐strand conformation polymorphism analysis was used to scan exon 17 of the c‐kit gene for mutations in genomic DNA of all family members and somatic DNA extracted from skin of the eldest affected sibling, the proband. No mutations were found in exon 17 in either genomic DNA of all family members or somatic DNA of the proband. Patients with UP have been shown to possess somatic mutations of the c‐kit gene. However, this locus has been excluded as playing a part in the three siblings examined here in whom a second gene locus must be determining their UP. Therefore, this study emphasizes genetic heterogeneity in UP. Future study to identify primary molecular determinants of UP should include affected sib‐pair studies.

Immunocytochemical localization of carbonic anhydrase isozymes I, II, and III in rat skeletal muscle.

Specific antisera were raised against the three carbonic anhydrase (CA) isozymes, CAI, CAII, and CAIII, and were used to determine the fiber distribution of these isozymes in skeletal muscle. Fiber types were determined by ATPase staining, and the CA isozymes were detected using a peroxidase-anti-peroxidase (PAP) technique. All three isozymes were present in type I fibers; CAII and CAIII were exclusive to these fibers, and CAI were also present in some small type 2A fibers.

Mutation detection in long QT syndrome: a comprehensive set of primers and PCR conditions

Editor—Long QT syndrome (LQTS) is an inherited disorder which produces arrhythmia and sudden death. The only presymptomatic indication of the disorder is an extended QT interval, in excess of 460 ms.1 However, those with LQTS do not always show this prolongation of QT. There are dominantly and recessively inherited forms of the disease, Romano-Ward syndrome and Jervell Lange-Nielsen syndrome, respectively, the latter also exhibiting severe sensorineural deafness.2-4 About half the familial cases of LQT are known to be caused by mutations in five ion channel or channel associated genes, with over 90% being accounted for by KCNQ1 and HERG , both of which code for potassium channels.5 The sodium channel gene SCN5A is responsible for about 8% of cases with a known gene mutation, while KCNE1 and KCNE2 , which code for proteins that associate with KCNQ1 and HERG respectively, are mutated in 1-2%.5 Mutations in KCNQ1 can produce both dominant and recessive forms of the disease, depending on the nature of the mutation.6-12 HERG and SCN5A mutations are dominant, while those in KCNE1 and KCNE2 are recessive.13-16There are, however, exceptions to these rules.17 18 Mutations have been identified throughout the genes,19 20although analysis of both HERG and SCN5A has tended to be concentrated on the pore regions owing to the substantial number of exons in both genes. The initial publications on KCNQ1 (formerly KVLQT1 ) also analysed the pore and surrounding regions, although several groups have now produced primers that cover the entire gene.19 21 The mutation analysis has been by PCR followed by SSCP, and although laborious, this is still the most commonly used method. Investigation of all the genes has not been possible, mainly because the exact sequence of the PCR fragments amplified by the …

Immunoassay of carbonic anhydrase III in rat tissues

Relatively high levels (i.e., up to 1% wet muscle wt) of a low-activity sulfonamide-resistant carbonic anhydrase isozyme has been reported from skeletal muscle of chicken, cat and sheep [ 11, ox [2], rabbit [3], mouse [4], human [8,9] and gorilla [7]. It has been accepted that these isozymes are all homologous forms of an enzyme designated CA111 and essentially confined to skeletal muscle. Nevertheless, trace levels of CA111 have also been detected immunologically in sheep lung 141, rabbit liver [4], and human liver, smooth muscle, lung and cardiac muscle [ 81. Assay of carbonic anhydrase III was carried out as described for human CA111 [8]. Antiserum was prepared in New Zealand white rabbits using 3 sequential weekly injections of 1 mg CA111 in Freund’s complete adjuvant, followed by an i.v. injection of 1 mg rat CA111 in 0.005 M phosphate buffer (pH 7.0). Tissue extracts (20%, w/v) were prepared in distilled water and spun at 20 000 X g for 15 mm. Extracts from soleus, anterior tibialis (AT), extensor digitorum longus (EDL), prostate, white fat and liver were made as above.

SEXUAL DIFFERENTIATION OF RAT LIVER CARBONIC ANHYDRASE III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.

Hormonal Control of Carbonic Anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III (CA III) in the livers of adult male rats was found to be approximately 30 times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver CA III concentrations that could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomized female rats induced about a 5-fold increase in liver CA III concentration. Immunoprecipitational analysis of the products of liver mRNA translation in vitro with antiserum specific for CA III showed that hormonal control of the levels of CA III in rat liver is mediated by changes in the amount of translatable CA III mRNA. Marked changes in liver CA III concentrations were also observed in developing and aging male rats. Different control mechanisms appear to operate in mouse and man.

Absence of linkage of Noonan syndrome to the neurofibromatosis type 1 locus.

Eleven families with Noonan syndrome in either two or three generations have been identified. Following the reports of subjects with features of both Noonan syndrome and neurofibromatosis type 1, these pedigrees have been studied using a number of probes at the neurofibromatosis type 1 locus (17q11). A significantly negative lod score was obtained with the intragenic probe NF1-C2, suggesting that the genes for Noonan syndrome and neurofibromatosis type 1 are not contiguous.

Radioimmunoassay of human muscle carbonic anhydrase III in dystrophic states.

A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4 microgram/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than levels in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease.