Exclusion of the expansion of CAG/CTG repeats at thirteen loci on chromosome 12 as a candidate genetic mutation in scapuloperoneal spinal muscular atrophy with anticipation

Scapuloperoneal spinal muscular atrophy (SPSMA) is a neuromuscular disorder characterized by weakness in the distribution of shoulder girdle and peroneal muscles. We have previously described a large New England kindred with autosomal dominant SPSMA and have subsequently linked this family trait to 12q24.1-q24.31. In this family, disease expression becomes more severe and progressive in successive generations, suggesting genetic anticipation. Accordingly, we have investigated the thirteen known CAG/CTG repeat loci on chromosome 12 that could be tested by using the polymerase chain reaction as candidate genetic mutations in SPSMA. None of these loci is expanded. Received: 12 June 1996 / Revised: 24 January 1997     

Glutathione contributes to plant defence against parasitic cyst nematodes

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host's roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (l -γ-glutamyl-l -cysteinyl-glycine) in Arabidopsis thaliana on infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All glutathione-deficient mutants exhibited impaired activation of defence marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant resistance to CN infection and the production of camalexin on nematode infection. These results suggest that glutathione levels affect plant resistance to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defence against infection.     

Benzimidazole inhibitors of the protein kinase CHK2: Clarification of the binding mode by flexible side chain docking and protein–ligand crystallography

Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.     

Thidiazuron induced somatic embryogenesis and plant regeneration in Capsicum annuum

An efficient protocol of direct somatic embryogenesis (without involving intermediate callus) has been developed from stem segments and shoot tips of Capsicum annuum L. Explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). Among the various concentration of TDZ tested, 0.5 μM was proved to be best for induction of somatic embryos. Induction, maturation and germination were achieved on the same medium. The shoots developed from somatic embryos were transferred for rooting to MS medium supplemented with indole-3-butyric acid (IBA). All the regenerated plants with 85 % survival rate were normal with respect to morphology and growth characteristics.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Progress in Wheat Resistance to Spot Blotch in Bangladesh

Spot blotch, caused by Cochliobolus sativus, is considered one of the most destructive diseases of wheat (Triticum aestivum) in the warm areas of South Asia. Over the past 20 years, wheat breeding efforts in the region have improved spot blotch resistance in susceptible commercial cultivars. This study assessed resistance and spot blotch‐induced yield losses in newly released wheat cultivars developed in Bangladesh since the release of the landmark wheat variety ‘Kanchan’. Replicated field studies were conducted during the 2003 and 2004 wheat seasons at two sites: a farmer's field and a research station in a warm region of Bangladesh where spot blotch has been a serious problem. Spot blotch affected 60% of the crop and caused yield losses of from 2% to 22%. Disease severity and disease‐induced grain yield reductions were less in wheat genotypes developed since 1983, with a corresponding trend towards higher yield in newly developed varieties. The level of resistance to spot blotch in the new cultivars and advanced breeding lines represents considerable progress in breeding for resistance over the past two decades.     

Serogroup,Virulence, and Genetic Traits of Vibrio parahaemolyticus in the Estuarine Ecosystem of Bangladesh

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., “clonal cluster,” as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.Vibrio parahaemolyticus, a halophilic bacterium, is a causative agent of seafood-related gastroenteritis worldwide (5, 13, 41) and one of the major causes of seafood-associated gastroenteritis in the United States, Asia, Europe, and countries where sporadic cases and outbreaks occur regularly (12, 13). The bacterium is prevalent in brackish and marine waters (43). Historically first identified as the causative agent of a gastroenteritis outbreak in Japan in 1950 (14), V. parahaemolyticus is now recognized as one of the most important food-borne pathogens in Asia, causing approximately half of food poisoning outbreaks in Taiwan, Japan, Vietnam, and Southeast Asian countries.The gene encoding the thermostable direct hemolysin (TDH)—manifested as beta-hemolysis when V. parahaemolyticus is plated onto Wagatsuma blood agar (43), i.e., the Kanagawa phenomenon (KP)—has been shown to be present in more than 90% of clinical strains and less than 1% of environmental strains (31, 39). Some strains also possess the gene trh, encoding the TDH-related hemolysin (TRH), or both tdh and trh (18, 43). Another gene, the thermolabile hemolysin gene (tlh), was reported to be present in V. parahaemolyticus (36) and subsequently in all V. parahaemolyticus strains tested (38).V. parahaemolyticus gastroenteritis is a multiserogroup affliction, with at least 13 O serogroups and 71 K serotypes detected (19, 42). In 1996, serogroup O3:K6 was first reported from diarrhea patients in Kolkata, India (32), and subsequently worldwide, as an increasing incidence of gastroenteritis caused by the serogroup O3:K6 was reported in many countries (41). Rapid spreading of serogroup O3:K6 infections in Asia (27, 32), and subsequently in the United States (12), Africa (3), Europe (25), and Latin America (15), indicated its potential as a pandemic pathogen (34, 43). In addition, V. parahaemolyticus serogroup O3:K6 possesses the group-specific (GS) gene sequence in the toxRS operon and ORF8, of the 10 known open reading frames (ORFs) of the O3:K6-specific filamentous phage f237. The GS gene and ORF8 provide genetic markers distinguishing O3:K6 from other serogroups (27, 29). Recent studies have shown O4:K68, O1:K25, O1:K26, O1:K untypeable (O1:KUT), and O3:K46 serogroups to share genetic markers specific for the pandemic serogroup O3:K6 (7, 10, 27, 34, 41). The non-O3:K6 serogroups with pandemic traits are increasingly found worldwide, and therefore, their pandemic potential cannot be ruled out.In Bangladesh, strains of different serogroups having genetic markers for the serogroup O3:K6 of V. parahaemolyticus were reported to have been isolated from hospitalized gastroenteritis patients in Dhaka (7). A systematic surveillance of the coastal areas bordering the Bay of Bengal where diarrheal disease is endemic (1) has not been done. This study, the first of its kind, was undertaken to investigate virulence potential, as well as phenotypic and genotypic traits of V. parahaemolyticus strains occurring in the estuarine ecosystem of Bangladesh.     

Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models

Background

The mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well.

Methods

Female DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR.

Results

Plasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver.

Conclusion

Down-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.     

Induction of chromosomal aberrations and sister chromatid exchanges by chlormadinone acetate in human lymphocytes: a possible role of reactive oxygen species

Genotoxicity study of a synthetic progestin chlormadinone acetate (CMA) was carried out in human lymphocytes using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) as parameter. Effect of CMA was studied at 10, 20, 30 and 40 microM. CMA was genotoxic at 30 and 40 microM. With a view to study the possible mechanism of genotoxicity of CMA, superoxide dismutase (SOD) and catalase (CAT) were used separately and in combination along with the CMA (40 microM) at different doses. SOD treatment increased CAs and SCEs at both the doses. CAT treatment decreased the frequencies of CAs and SCEs in both, separately and in combination with SOD, suggesting a possible role of reactive oxygen species for the genotoxic damage.