Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.     

Post-thyroidectomy hypoparathyroidism

The incidence of hypoparathyroidism following thyroid surgery in the series reported was 7.8 per cent. Temporary hypoparathyroidism was present in 15 patients (4.6%) and permanent hypoparathyroidism in 10 (3.2%). Persistently low levels of serum calcium and high levels of serum phosphorus at six to 10 weeks after thyroid operation in patients with temporary hypoparathyroidism, with no tendency toward normalization, suggests permanent hypoparathyroidism. Serum calcium below 5 mg per cent suggests permanent hypoparathyroidism.     

Correlation of PPD and BCG-induced leukocyte migration inhibition, delayed cutaneous hypersensitivity, lymphocyte transformation in vitro and humoral antibodies to PPD in man

A laboratory colony of O. moubata was maintained entirely by feeding on the exposed membranes of embryonated hen eggs at the 17-19th day stage of incubation at 37 degrees C. There was no loss of either vigour or fecundity. The method gave markedly superior results to those obtained by feeding on rabbits, rats, mice and guinea-pigs. More convenient and economical than any alternative, the method has valuable advantages in its applicability to a wide range of pathogens.     

Pneumoperitoneum: importance of right upper quadrant features

Supine abdominal radiographs of 59 patients with documented pneumoperitoneum were evaluated for findings of pneumoperitoneum. The commonly described signs were seen in only 17%. Localized intraperitoneal air in the right upper quadrant was the most frequent finding, seen in 49% of patients. Identification of these collections of air is dependent upon knowledge of the anatomy of the perihepatic spaces.     

Immunogenicity and conformational properties of an N-linked glycosylated peptide epitope of human T-lymphotropic virus type 1 (HTLV-I)

The identification and characterization of epitopes of human T-lymphotropic virus type 1 (HTLV-I), which elicit an effective humoral or cell-mediated immune response, remains a central obstacle to the development of a peptide-based vaccine against the virus infection. The objective of the studies presented here was to examine the influence of N-linked glycosylation on peptide structure and immunogenicity. We engineered the 233-253 sequence of gp46 of HTLV-I to contain an N-acetylglucosamine (GlcNAc) residue at Asn244. Secondary structure prediction using computer algorithms indicated that this peptide may contain a beta-turn at residues 242-246. Recent work with model glycopeptides suggests that beta-turn conformation in peptides may be induced, and probably is stabilized, by the presence of even a single sugar residue. In the present study, the structures of the 233-253 peptide, SC1, and the 233-253(Asn244-GlcNAc) glycopeptide, SC2, were determined. Similar conformation was exhibited by both the glycosylated and nonglycosylated peptide displaying a beta-turn at residues 243-246 and extended-chain structure at the peptide/glycopeptide termini. Both peptides were engineered into chimeric constructs with a promiscuous T-cell epitope from measles virus and were used as immunogens in rabbits. Both chimeric peptides were highly immunogenic in rabbits, producing high-titered antibodies as early as primary + three weeks. The antibodies generated against either construct were able to bind to whole virus (ELISA) and to gp46 (radioimmunoprecipitation assay). Additionally, human sera of individuals known to be positive for HTLV-I recognized both the glycosylated and nonglycosylated constructs. It appears that the 233-253 peptide is able to adopt a conformation that mimics the structure in native gp46, and addition of a GlcNAc residue at Asn244 does not affect the conformational preference or stability of this construct; nor does glycosylation alter immunogenicity but instead appears to enhance immune recognition.     

The risk of infective thyroiditis in nodular goitres

Infective thyroiditis which was more common in the pre-antibiotic era is now rare in developed countries but continues to occur in less developed areas. Over a 10-year period in Zaria, Nigeria which is situated in an endemic goitre region, nine such patients were treated. All were females, 30-50 years of age and had pre-existing, long-standing multinodular goitres. During the period, 84 patients (including the nine patients) with endemic multinodular goitres were treated, giving an infective rate of 10.7% in these goitres. Treatment was by adequate incision and drainage in all patients with suppuration. Staphylococcus aureus was the commonest organism cultured and Streptococcus Pyogenes and Pseudomonas were the infective agents in a minority. Only one patient required thyroidectomy later, the goitre having disappeared in the other patients. One patient developed hypothyroidism requiring replacement therapy with thyroxine. Thyroid abscess remains an ever present risk in endemic multinodular goitres in our environment. Early presentation after onset of pain, prompt administration of appropriate antibiotics and thyroidectomy soon after is likely to reduce the risk of abscess formation in these goitres.     

Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes

Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 +/- 3% to 14 +/- 2%. Return of contraction was observed in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased approximately 2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 micromol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic acid, and the anion channel inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, all of which also abrogated protection. ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor L-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection. Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.