Comparative study in the Ames test of benzo[a]pyrene and 2-aminoanthracene metabolic activation using rat hepatic S9 and hepatocytes following in vivo or in vitro induction

We studied the replacement of hepatic S9 with in vivo and in vitro induced hepatocytes as a metabolic activation system with the aim of broadening the possibilities of mutagenic assays. Rats were pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), 3-methylcholanthrene (MC) and a combination of BNF and PB (BNF + PB). Mutagenic activation of benzo[a]pyrene (BP) and 2-aminoanthracene (2AA) by hepatic S9 and hepatocytes was determined in the Ames test. Primary rat hepatocytes were used for in vitro induction and were used as the activating system in the Ames test. In vivo BNF treatment greatly increased the metabolic activation capacity of hepatic S9 and hepatocytes towards BP. With regard to 2AA activation, S9 and hepatocytes showed different BNF induction profiles. PB treatment reduced the mutagenicity of both compounds. Although ethoxyresorufin O-dealkylase (EROD) activity of S9 from BNF + PB-treated animals was almost 30-fold greater than the control, its effectiveness in activation of 2AA was below the control level. A large part of the EROD activity of control cells was lost during culture, together with the ability to activate 2AA, however, 72 h of MC induction increased EROD activity to 200-fold of the control, which corresponds to 28% of that of in vivo induced hepatocytes. The mutagenic potential of BP activated by in vitro induced hepatocytes was 10-fold above the control, which is 47% of the mutagenicity detected following in vivo induction. In vitro induced hepatocytes increased 2AA mutagenicity to 14.6-fold over the control, which corresponds to 68% of in vivo induction. Our results suggest that primary culture of hepatocytes provides a useful model for the study of the role of metabolic activation processes concerning enzyme activity of cytochromes P450 and other metabolic enzymes and induction profiles of different inducers.     

Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.     

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. .     

Polymorphic membrane protein H has evolved in parallel with the three disease-causing groups of Chlamydia trachomatis

Chlamydia trachomatis is a human pathogen causing trachoma, urogenital disease, and lymphogranuloma venereum (LGV). A family of nine polymorphic membrane protein genes (pmpA to pmpI), resembling autotransporter proteins, has recently been discovered in C. trachomatis. pmp genes are large and predicted to be outer membrane proteins. We hypothesized that they would contain useful nucleotide sequence variability for epidemiologic studies. Since sequence information is available only for serovars D and L2, we sought to determine the amount of diversity within an individual pmp gene among serovars. We used restriction fragment length polymorphism (RFLP) analysis as a primary screen to assess the amount of sequence divergence among the pmp genes for serovars A to L3 of C. trachomatis. RFLP analysis showed little variation for some of the genes, such as pmpA, but substantial variation in others, such as pmpI. pmpH and pmpE yielded RFLP patterns that clustered the 15 serovars into ocular, urogenital, and LGV groups, and both proteins have been localized to the outer membrane. Therefore, we chose to sequence pmpE, pmpH, and pmpI from each of the 15 serovars. Evolutionary analysis showed three distinct divergence patterns. PmpI was least variable, resulting in an ambiguous evolutionary pattern. PmpE showed a high degree of diversity in the ocular strains compared to the other strains. Finally, the evolution of PmpH shows three groups that reflect disease groups, suggesting this protein may play a role in pathogenesis.     

A Quantitative Genetic Analysis of Locomotor Activity in CXB Recombinant Inbred Mice

Recent studies have identified genes that influence the length of the circadian period maintained by mice housed under constant lighting conditions. However, a less studied circadian activity variable is the amplitude of daily oscillations in locomotor activity. This parameter reflects spontaneous activity exhibited under standard lighting and housing conditions and, therefore, differs conceptually from assessments of exploratory or open-field activity, voluntary wheel-running, or circadian period during exposure to constant light or constant darkness conditions. We recently observed a greater daily amplitude of oscillation in spontaneous locomotor activity in C57BL/6 mice compared to BALB/cBy mice. To identify genetic loci with potential linkage to circadian variation in the amount of locomotor activity, we measured the spontaneous activity of 13 CXB recombinant inbred (RI) strains of mice. The probability density distributions of locomotor activity phenotypes for the 13 CXB RI strains were consistent with the presence of a low number of major quantitative trait loci affecting this trait. Regions of chromosomes 3, 8, 12, 13, and 19 showed provisional linkage to strain variation in locomotor activity. Probabilities of linkage were not sufficient for declaration of an activity-related quantitative trait locus but were sufficient to warrant further analysis either with additional RI strains or with F₂ panels.     

Transmission disequilibrium tests confirm the link between DRD4 gene polymorphism and infant attachment.

Following up the results of a previous population association study (Lakatos et al. [2000: Mol Psychiatry 5:633-637; Lakatos et al. [2002: Mol Psychiatry 7:27-31]) by analyses based on parental genetic data confirmed the link between infant attachment and the dopamine D4 receptor (DRD4) gene. Extended transmission disequilibrium tests (ETDT) were performed to determine whether biased transmission of exon III 48 basepair repeat alleles occurred to infants displaying disorganized and secure attachment behavior with their mothers. The overall allele-wise TDTs were significant for both groups (P = 0.038 and 0.020, respectively): a trend for preferential transmission of the seven-repeat allele to disorganized infants was observed (TDT(chi)(2) = 3.27, df = 1, P = 0.071), and there was a significant non-transmission of the same allele to securely attached infants (TDT(chi)(2) = 6.00, df = 1, P = 0.014). Analysis of haplotypes of the exon III repeat and the -521 C/T promoter polymorphisms in family trios showed that the transmission bias in the larger secure group was due to the low-rate transmission of the T.7 haplotype containing both the seven-repeat and the -521 T alleles (TDT(chi)(2) = 4.46, df = 1, P = 0.035). This suggests that not carrying the T.7 haplotype of the DRD4 gene may act as a resilience factor in the optimal development of early attachment.     

Malignancy may adversely influence the quality and behaviour of oocytes

A case series of eight cycles of in-vitro fertilization (IVF) in five women diagnosed with malignant disorders is presented. These patients chose to defer definitive treatment for a chance for preservation of potential fertility. The response of these patients to ovarian stimulation, and the outcome, was compared with 17 IVF cycles in 12 age- matched patients with isolated tubal infertility. An apparent adverse influence of malignant disease on the quality and behaviour of oocytes was observed. Despite a comparable total number of oocytes per cycle in the two groups, a significantly reduced percentage of mature oocytes was retrieved per cycle from patients with malignant diseases. The oocytes from patients with malignant disorders were of a poorer quality and exhibited a significantly impaired fertilization rate compared to the controls. We propose that neoplastic processes, irrespective of the site or cell of origin, may have a detrimental impact on the biology of oocytes, an effect akin to that seen on spermatozoa in men with certain malignancies.      

Prevalence of Antibodies to Hepatitis E Virus in Veterinarians Working with Swine and in Normal Blood Donors in the United States and Other Countries

Hepatitis E virus (HEV) is endemic in many developing and some industrialized countries. It has been hypothesized that animals may be the source of infection. The recent identification of swine HEV in U.S. pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis. To assess the potential risk of zoonotic HEV infection, we tested a total of 468 veterinarians working with swine (including 389 U.S. swine veterinarians) and 400 normal U.S. blood donors for immunoglobulin G anti-HEV. Recombinant capsid antigens from a U.S. strain of swine HEV and from a human HEV strain (Sar-55) were each used in an enzyme-linked immunosorbent assay. The anti-HEV prevalence assayed with the swine HEV antigen showed 97% concordance with that obtained with the human HEV antigen (kappa = 92%). Among the 295 swine veterinarians tested from the eight U.S. states (Minnesota, Indiana, Nebraska, Iowa, Illinois, Missouri, North Carolina, and Alabama) from which normal blood donor samples were available, 26% were positive with Sar-55 antigen and 23% were positive with swine HEV antigen. In contrast, 18% of the blood donors from the same eight U.S. states were positive with Sar-55 antigen and 17% were positive with swine HEV antigen. Swine veterinarians in the eight states were 1.51 times more likely when tested with swine HEV antigen (95% confidence interval, 1.03 to 2.20) and 1.46 times more likely when tested with Sar-55 antigen (95% confidence interval, 0.99 to 2.17) to be anti-HEV positive than normal blood donors. We did not find a difference in anti-HEV prevalence between veterinarians who reported having had a needle stick or cut and those who had not or between those who spent more time (> or = 80% of the time) and those who spent less time (< or = 20% of the time) working with pigs. Similarly, we did not find a difference in anti-HEV prevalence according to four job categories (academic, practicing, student, and industry veterinarians). There was a difference in anti-HEV prevalence in both swine veterinarians and blood donors among the eight selected states, with subjects from Minnesota six times more likely to be anti-HEV positive than those from Alabama. Age was not a factor in the observed differences from state to state. Anti-HEV prevalence in swine veterinarians and normal blood donors was age specific and paralleled increasing age. The results suggest that swine veterinarians may be at somewhat higher risk of HEV infection than are normal blood donors.