Genetic factors controlling inheritance of susceptibility to 1,2-dimethylhydrazine

Summary Reciprocal crosses were made between AKR/J, a 1,2-dimethylhydrazine (DMH)-resistant mouse strain, and SWR/J, a sensitive strain. The F1 hybrids were tested with DMH and methylazoxymethanol (MAM), two colon carcinogens. Either DMH (20 mg/kg body weight) or MAM (35 mg/kg body weight), a metabolic derivative of DMH, was injected weekly for 10 weeks. In each group of 35 mice, 10 were injected with tritiated thymidine (25 Ci) 1 week after the sixth injection of DMH and MAM for the evaluation of proliferative characteristics and the number of foci of dysplasia occuring in 325 m of distal colonic mucosa. At 27 weeks after the first injection of the carcinogen, the colons of remaining mice were opened longitudinally and the number of tumors enumerated. Compared with DMH-treated mice, the number of foci of dysplasia per mouse, the percentage of tumor-bearing mice, the number of tumors per animal, and the number of tumors per tumor-bearing animal induced by MAM were severalfold higher. This would suggest the presence of a gene(s) repressing metabolism of DMH to MAM. Moreover, differences in response to the carcinogens were observed between the sexes. In contrast to males, females treated with both DMH and MAM had significantly greater numbers of tumors per animal, tumors per tumor-bearing mice, and a greater proliferative response with extension of S-phase cells to the upper third and luminal surface of crypts. Among males, those with the X^AKR/Y^SWR heritage appeared more resistant than X^SWR/Y^AKR males, particularly in their response to MAM. A twofold difference in the number of foci of dysplasia per mouse, tumors per animal, and the number of tumors per tumor-bearing animals was seen. Analyses of the response to DMH and MAM by F1 reciprocal hybrids of the AKR and SWR strains have shown a complex inheritance pattern governing susceptibility to DMH. Resistance to the carcinogen is provided by at least two specific repressor genes, one governing metabolism of carcinogen from DMH to MAM, and the other controlled by gender. Genetic factors contributed by the AKR female appear to convey additional resistance to male progeny, suggesting more than one gender-related gene.Supported in part by CA 08748 from the National Cancer Institute and by CA 26674 from the National Cancer Institute through the National Large Bowel Cancer Project     

An association between variants in the IGF2 gene and Beckwith-Wiedemann syndrome: interaction between genotype and epigenotype

Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth disorder involving the deregulation of a number of genes, including IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. In sporadic BWS cases the majority of patients have epimutations in this region. Loss of imprinting of the IGF2 gene is frequently observed in BWS, as is reduced CDKN1C expression related to loss of maternal allele-specific methylation (LOM) of the differentially methylated region KvDMR1. The causes of epimutations are unknown, although recently an association with assisted reproductive technologies has been described. To date the only genetic mutations described in BWS are in the CDKN1C gene. In order to screen for other genetic predispositions to BWS, the conserved sequences between human and mouse differentially methylated regions (DMRs) of the IGF2 gene were analyzed for variants. Four single nucleotide polymorphisms (SNPs) were found in DMR0 (T123C, G358A, T382G and A402G) which occurred in three out of 16 possible haplotypes: TGTA, CATG and CAGA. DNA samples from a cohort of sporadic BWS patients and healthy controls were genotyped for the DMR0 SNPs. There was a significant increase in the frequency of the CAGA haplotype and a significant decrease in the frequency of the CATG haplotype in the patient cohort compared to controls. These associations were still significant in a BWS subgroup with KvDMR1 LOM, suggesting that the G allele at T382G SNP (CAGA haplotype) is associated with LOM at KvDMR1. This indicates either a genetic predisposition to LOM or interactions between genotype and epigenotype that impinge on the disease phenotype.     

Arterial endothelial function in a porcine model of early stage atherosclerotic vascular disease

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the United States and is projected to become the leading cause of mortality in the world. Atherosclerosis is the most important single factor contributing to this disease burden. In this study, we characterize relationships between endothelial dysfunction and vascular disease in an animal model of diet-induced, early-stage atherosclerotic vascular disease. We tested the hypothesis that hypercholesterolaemia induces vascular disease and impairs endothelium-dependent relaxation (EDR) in conduit arteries of adult male Yucatan pigs. Pigs were fed a normal fat (NF) or high fat cholesterol (HFC) diet for 20-24 weeks. Results indicate that, while the HFC diet did not alter EDR in femoral or brachial arteries, EDR was significantly decreased in both carotid and coronary arteries. Sudanophilic fatty streaks were significantly present in the abdominal aorta and common carotid artery. Histopathology revealed increased intima-media thickness (IMT) and foam cell accumulation in Stary Stage I-III lesions in the abdominal aorta, common carotid artery and femoral arteries. In the coronary arteries, the accumulation of foam cells in Stary Stage I and II lesions resulted in a trend for increased IMT. There was no evidence of vascular disease in the brachial arteries. These results indicate that early stages of CVD (Stary Stage I-III) precede decreases in EDR induced by HFC diet, because femoral arteries exhibited foam cell accumulation and an increased IMT but no change in endothelial function.     

Mitogen-activated Xenopus laevis lymphocytes produce a T-cell growth factor.

Mitogen-free and serum-free supernatants (SNs) from cultures of phytohaemagglutinin (PHA)-stimulated Xenopus splenocytes, co-stimulated thymocytes, induced proliferation of splenic and thymic lymphoblast and supported growth of alloreactive T-cell lines. These SNs had no effect on 'resting' splenocytes, as measured by uptake of tritiated thymidine ([3H]TdR). Growth-promoting activity was also detected in SNs of cultures containing alloreactive T-cell lines and either PHA or irradiated stimulator cells that expressed the original priming alloantigens. Thus, T lymphocytes appear to be involved in producing, as well as responding to, a Xenopus T-cell growth factor (TCGF). TCGF activity could be absorbed from these active SNs with PHA-activated splenic blasts. No functional cross-reactivity among different mammalian interleukin-2 (IL-2) and Xenopus TCGF preparations was detected.     

A randomized controlled trial of an information booklet for hypertensive patients in general practice

A randomized controlled trial of an information and medical record booklet designed to improve patient understanding and participation in the management of hypertension was conducted in six inner London general practices. After one year there were no significant differences between the group who had received the booklets and the control group in mean systolic or diastolic blood pressure, but the study group scored significantly higher on knowledge about hypertension and its management. However, the difference between the two groups was small, possibly because both groups started with a high level of understanding about hypertension and its management. In addition, the mean diastolic blood pressure in the control group showed that the treatment provided was already satisfactory, and that there was little need for improvement. Nevertheless, the information booklet evaluated in this study provides health professionals with a highly acceptable method of informing the patient about hypertension and its management and could be used both in hospital and general practice.     

Unconventional therapies in asthma: an overview

Acupuncture, homoeopathy, mind-body therapies, and nutritional, herbal, and environmental medicine have all been used in the management of patients with asthma. This paper reviews the evidence base for the use of these unconventional or complementary therapics. Although there is a paucity of large randomized, controlled trials in this area, there is sufficient evidence to suggest that many of these therapies can produce objective and subjective benefit in selected groups of patients. In view of the increasing popularity of complementary medicine among patients and general practitioners, there is now an urgent need for high-quality research to determine how, or whether, these therapies may be interwoven with the more orthodox treatments currently available.     

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Vitamin C-induced loss of redox-dependent viability in lung microvascular endothelial cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.