Chronic arsenic toxicity: studies in West Bengal, India.

Chronic arsenic toxicity (arsenicosis) as a result of drinking arsenic-contaminated groundwater is a major environmental health hazard throughout the world, including India. A lot of research on health effects, including genotoxic effect of chronic arsenic toxicity in humans, have been carried out in West Bengal during the last 2 decades. A review of literature including information available from West Bengal has been made to characterize the problem. Scientific journals, monographs, and proceedings of conferences with regard to human health effects, including genotoxicity, of chronic arsenic toxicity have been reviewed. Pigmentation and keratosis are the specific skin diseases characteristic of chronic arsenic toxicity. However, in West Bengal, it was found to produce various systemic manifestations, such as chronic lung disease, characterized by chronic bronchitis, chronic obstructive and/or restrictive pulmonary disease, and bronchiectasis; liver diseases, such as non cirrhotic portal fibrosis; polyneuropathy; peripheral vascular disease; hypertension; nonpitting edema of feet/hands; conjunctival congestion; weakness; and anemia. High concentrations of arsenic, greater than or equal to 200 µg/L, during pregnancy were found to be associated with a sixfold increased risk for stillbirth. Cancers of skin, lung, and urinary bladder are the important cancers associated with this toxicity. Of the various genotoxic effects of arsenic in humans, chromosomal aberration and increased frequency of micronuclei in different cell types have been found to be significant. Various probable mechanisms have been incriminated to cause DNA damage because of chronic arsenic toxicity. The results of the study in West Bengal suggest that deficiency in DNA repair capacity, perturbation of methylation of promoter region of p53 and p16 genes, and genomic methylation alteration may be involved in arsenic-induced disease manifestation in humans. P53 polymorphism has been found to be associated with increased occurrence of arsenic-induced keratosis. Of the various genes involved in the regulation of arsenic metabolism, single-nucleotide polymorphisms of purine nucleoside phosphorylase, in one study, showed increased occurrence of arsenicosis.

Haplotype analysis at the FRAXA locus in an Indian population.

The FRAXA locus is flanked by three polymorphic STR markers DXS548, FRAXAC1, and FRAXAC2. Allele frequencies of these markers were determined on a population representing the eastern part of India comprising of 69 normal controls and 69 unrelated subjects with mental retardation, among whom 21 were fragile X patients. These frequencies were compared with published data on other Indian population and the major populations of the world. The allele and haplotype distribution of the studied population were significantly different in some respects from the major populations of the world. The increase of heterozygosities in fragile X samples (DXS548 67.5%, FRAXAC1 63.5%, FRAXAC2 68.5%) relative to the controls (DXS548 63.3%, FRAXAC1 51.0%, FRAXAC2 67.2%) suggests a multimodal distribution of fragile X associated alleles. Haplotype analyses with DXS548 and FRAXAC1 markers revealed that haplotype distribution in the normal controls and fragile X groups were significantly different, suggesting a weak founder effect.

e19a2 BCR-ABL fusion transcript in typical chronic myeloid leukaemia: a report of two cases.

This report describes two patients with chronic myeloid leukaemia (CML): one of them developed accelerated phase CML and died 8 years after diagnosis and the other is at the chronic phase. Sequence analysis of reverse transcription-polymerase chain reaction products showed the presence of BCR-ABL fusion transcript e19a2. This finding suggests that CML carrying mu-BCR breakpoint may exhibit a clinical course similar to typical CML.

Syndromic approach for determination of reproductive tract infections among adolescent girls.

The present study shows overall prevalence (64%) of reproductive tract infection among adolescent girls, based on self-perceived symptoms. Mean age of respondents were found to be 17.8 +/- 0.82 years and mean age at marriage and mean age at first pregnancy were 17.2 years and 17.5 years respectively; 35.35% of girls in the present study were married. In addition, no significant difference was observed between unmarried (60.10%) and married (71.17%) reproductive tract infection groups. Moreover, no significant association was present in prevalence of reproductive tract infection between the Muslim (67%) and the Hindu (60%). Highest prevalence (84.06%) of reproductive tract infection was observed among illiterate girls and with improvement of educational status there was decrease in the prevalence and the association was found highly significant. Significantly, higher prevalence (72%) was observed among members of family size 7 and above.

Glutathione S-transferase M1 and T1 null genotype frequency in chronic myeloid leukaemia.

Polymorphisms associated with genes coding for glutathione S-transferase enzymes are known to influence metabolism of different carcinogens and have been associated with incidence of various types of cancer. We have determined the GST M1 and GST T1 'null' genotype frequency in 81 patients with chronic myeloid leukaemia (CML) and 123 racially and geographically matched control individuals by multiplex polymerase chain reaction (PCR). GST M1 null genotype frequencies in CML and controls were 28.4% and 27.7%, respectively. GST T1 null genotype frequencies in CML and controls were 19.8% and 7.3%, respectively. The GST T1 null genotype frequency in CML patients is significantly different from that in controls (odds ratio (OR) 3.12, 95% confidence interval (CI) 1.3-7.45, P=0.008).

Induction of apoptosis by Phenothiazine derivatives in V79 cells.

Phenothiazine derivatives chlorpromazine (cpz) and trifluoperazine (tfp) were found to induce apoptosis, abnormal cell cycle and expression of p53 in Chinese hamster lung fibroblast V79 cells. Both the drugs can induce apoptosis when cells are treated with drug at a concentration of 10 microg/ml within 4 h, as detected by propidium iodide staining and DNA fragmentation analysis. Flow cytometric analysis revealed that the apoptotic response is mediated by a loss of G(1) population of cells. In Western blot analysis, p21 is induced and p53 is accompanied by additional bands. Also indirect immunolabeling of single cells revealed that p21 is accumulated from cytoplasm into nucleus after the drug treatment and the intensities of p53 increased. Our findings demonstrate for the first time that phenothiazine derivatives, in addition to their cytotoxic effects, could induce apoptosis, an observation that has important clinical implications.

Fragile X syndrome in Calcutta, India.

Fragile-X-linked mental retardation usually results from amplification of the CGG repeat in the 5' untranslated region of the FMR1 gene. To assess the extent of variation of the CGG repeat in the population from the eastern region of India we studied 98 mentally retarded individuals living in and around Calcutta and identified 21 distinct alleles ranging in size from 8 to 44 CGG repeats. A repeat size of 28 was the most frequent; this value is different from the most frequent repeat size found in other studies, indicating a racial or ethnic variation. Patients with the clinical features of the syndrome have been found to carry expanded CGG repeats. Thus, it can be inferred that the expansion of CGG repeats may be a frequent cause of the syndrome in our population.

Fragile X syndrome a case report of a family.

Fragile X syndrome is the most common of the inherited disorders causing mental retardation. This disorder results from an abnormal expansion in (CGG)n in repeat found in the coding sequence of the FMRI gene, located at Xq 27.3. Previously it was detected by Karyotyping. With the advent of Molecular Biology PCR, has become the best method in the diagnosis of this disorder. This is a case report of a family with this disorder detected by PCR.

Variable severity of beta-thalassemia patients of eastern India: effect of alpha-thalassemia and xmnI polymorphism.

Sixty-four thalassemia and E-beta thalassemia patients were studied for factors that modulate the severity of the disease; i.e., mutation of beta-globin gene, presence of alpha-deletion, and presence of an XmnI site at the -158 position of the Gy gene. Presence of alpha-deletion and/or homozygosity for the XmnI site was in general associated with less-severe disease. About 12% of the patients harbored single alpha-gene deletion, and the gene frequency of the XmnI polymophism in these patients is 0.48.

ras gene mutations in oral cancer in eastern India.

Oral tumor specimens (n = 50) from eastern Indian population were studied for the presence of mutations in the H-, K- and N-ras genes using selective oligodeoxynucleotide hybridization and restriction fragment length polymorphism analysis of polymerase chain reaction-amplified products. Mutations in H- and K-ras genes were observed at a frequency of 28 and 33%, respectively, whereas no N-ras mutation was noticed.

Major beta-globin gene mutations in eastern India and their associated haplotypes.

324 alleles of the beta-globin gene from unrelated thalassaemia patients native to the eastern region of India (mainly from the state of West Bengal) were analysed for beta-globin gene mutations by the amplification refractory mutation system (ARMS). The major mutations that were detected are IVS-1 pos 5 (G-C), codon 26 (G-A) and codon 30 (G-C) with frequencies of 0.45, 0.33 and 0.05, respectively. Haplotype analysis revealed a very strong linkage disequilibrium of IVS-1 pos 5 (G-C) with one particular haplotype. HbE was found to be associated with two major haplotypes. Codon 30 (G-C) was associated with a haplotype that is the same as that found in the African population. Haplotype associated with codon 8/9 (+G) was the same as that found in northwest India. These findings have implications for the use of molecular diagnosis for genetic counselling and prenatal diagnosis of beta-thalassaemia in this region.

Report of a hitherto unreported sequence present and expressed in a cancer cell line.

During the amplification of a 202 base pair (bp) fragment around the 61st codon of the N-ras gene, an extra band of about 150 bp in length was observed when genomic DNA from a human epidermoid carcinoma cell line was used as template. This fragment was cloned and sequenced. However, this sequence was not found in the databank. RT-PCR experiments indicated that the sequence is expressed in the cells about 8 h after serum induction. The relevant RNA hybridized to one strand of the sequence but not to the other.

Sequence comparison of the chromosomal regions encompassing the internalin C genes (inlC) of Listeria monocytogenes and L. ivanovii.

We have recently cloned and characterized the inlC gene of Listeria monocytogenes which belongs to the listerial internalin multigene family and codes for a 30-kDa secreted protein containing five consecutive leucine-rich repeats. Here, we show that in L. monocytogenes inlC is located between the rplS gene (encoding the 50S ribosomal protein L19), and the infC gene (encoding the translation initiation factor 3). By direct and inverse polymerase chain reactions (PCR), we cloned a 5.4-kb region containing a homologous gene (termed i-inlC) from L. ivanovii, the other pathogenic member of the genus Listeria. In this microorganism, the i-inlC gene is preceded by another internalin gene, i-inlD, which seems to be specific for L. ivanovii, as this gene could not be detected in L. monocytogenes by Southern hybridization with an i-inlD gene probe. The i-inlD gene also encodes a small secretory internalin (i-InlD), which shares extended homology with (i-)InlC. Upstream of i-inlD are genes for 23S rRNA and 5S rRNA, and two tRNA genes [Asn-tDNA (GTT) and Thr-tDNA(GTT)]. The 3' terminus of the Thr-tRNA gene appears to be the site of an insertion of a genetic element including i-inlC and i-inlD. A putative transcriptional regulator gene, the product of which contains the TetR family signature, is located downstream of i-inlC. This chromosomal position of the two inlC genes on their respective chromosomes may be due to horizontal transfer of this gene. Transcription of i-inlC and i-inlD is strictly dependent on the transcriptional activator PrfA, which regulates transcription of most of the known virulence genes (including inlC) of L. monocytogenes and of L. ivanovii.

Cholera vaccine: developmental strategies and problems.

Over a hundred years have elapsed since Vibrio cholerae, the etiological agent for the disease cholera, was discovered by Robert Koch. Ever since then serious efforts have been made to develop prophylactic measures to combat the disease without much success. Seven pandemics have so far been reported and cholera still remains a public health problem in developing countries. Several strategies have been adopted to develop vaccines against the disease and many of these vaccines have undergone field trials. During the last two decades, an enormous amount of information has accumulated regarding the organism V. cholerae, its virulence factors, including cholera toxin, and the molecular basis of its pathogenicity. In recent years, with the advent of recombinant DNA technology and major breakthroughs in molecular biology and immunology, a new dimension has been given to the design of vaccine strains. The second generation live oral vaccines will perhaps soon replace the long-used first generation parenterally administered killed whole cell vaccines which offered protection for not more than three months. All the recombinant vaccines tested so far produced adverse reactions in volunteers, although they provided varying degrees of protection upto about one year of surveillance. Parallel to the trials of live oral vaccines, combination vaccines comprising killed whole cells and purified B subunit of cholera toxin was also tried. These vaccines had minimal side-effects but the efficacy was not upto expectations. From the failure of each vaccine strain, new information had emerged and improved strategies were adopted.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of strain viability and antigen dose on the use of attenuated mutants of Salmonella as vaccine carriers.

It is now accepted that oral killed typhoid vaccines are not effective at inducing protective anti-typhoid immunity. It is not known whether oral killed Salmonella can function as an effective carrier of other antigens to the immune system. In order to test this hypothesis, we immunized groups of mice with viable and non-viable preparations of aroA Salmonella dublin strain EL23 which codes for production of the binding subunit of the heat-labile enterotoxin of Escherichia coli (LT-B). Animals immunized orally with viable EL23 developed serum and mucosal anti-LT-B responses consistent with our previous findings. Significantly, mice immunized orally with ultraviolet-killed EL23 developed serum and mucosal antibody responses equivalent to those which developed in animals orally immunized with the same number of viable EL23. We extended these observations to include a number of methods of killing the organisms which may also preserve the ability of these strains to function as carriers. Our findings indicate that viability is not a requirement for use of a Salmonella strain as an immunological carrier. Moreover, our evidence indicates that bacteraemia and persistence in tissues are not necessary for oral priming, and therefore it may be best to dissociate the question of what makes the best live oral anti-typhoid vaccine from the question of what makes a good carrier of heterologous antigens.

Cytotoxic and genetic effects of X-irradiation of human cells in the presence of chlorpromazine.

Human epidermoid carcinoma cells (Hep-2) were X-irradiated in the presence of 5-10 micrograms/ml of chlorpromazine (CPZ). Survival of the cells decreased with increasing CPZ concentration. Lymphocytes from three normal volunteers exposed to X-irradiation in the presence of CPZ showed an increased frequency of dicentric and ring formation.

Recombinant derivative of a naturally occurring non-toxinogenic Vibrio cholerae 01 expressing the B subunit of cholera toxin: a potential oral vaccine strain.

A clinical isolate of Vibrio cholerae 01 was identified which did not possess the heat-labile (CT), the heat-stable (ST) or the zonula occludens (Zot) toxin genes. Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain. The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate. The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V. cholerae for the GM1 ganglioside binding sites in vivo. This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea (RITARD) model. This construct has an advantage over other live oral attenuated V. cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic.

Transformation of Vibrio cholerae by plasmid DNA.

The lack of an efficient transformation system in Vibrio cholerae was a handicap in the genetic manipulation of this important human pathogen. Since V. cholerae cells secrete DNases, this may interfere with the uptake of DNA. The present report describes the approaches taken for transforming V. cholerae cells with plasmid DNA, by overcoming this DNase barrier. The partial success of transforming DNase-negative mutants confirmed the role of DNase in the nontransformability of the wild-type cells. Successful transformation was carried out following removal of DNases from the periplasmic space. This was achieved by treating the cells with Mg2+ and Ca2+ ions to allow the DNase to be released, and then holding them under conditions where the remaining DNase activity was minimized before adding DNA to the competent cells. Transformation efficiencies of the order of 10(-5) per recipient cell were observed.

Nonrandom chromosome changes in methylnitrosourea (MNU) induced mouse T-cell lymphomas.

Since nonrandom chromosome changes in neoplastic cells have proven to be good indicators of the site of gene alterations related to transformation, the authors examined the chromosomes of T-cell lymphomas induced in RF/J strain mice with methylnitrosourea (MNU). All treated mice developed thymic lymphomas within 10 weeks of injection. Chromosomes of the thymus cells were examined at intervals before and during lymphoma development, as well as after they were passaged in syngeneic and in nude mice for periods up to 424 days. In preparations made directly from the thymus cells nonrandom numerical and structural alterations were found that involved the X, 3, 15, 4, 8, 12, 14 and 17. (Chromosomes showing alterations are listed in decreasing order of the frequency of their occurrence). In cells passaged in nude mice the chromosomes similarly altered were the 10, X, 3, 12, 6, 1, 4, 19, 15, 18 and 14. In tumor cells passaged in syngeneic mice most of the same chromosomes were involved but the order was 15, 14, X, 1, 5, 6, 3, 11 and 12. The X, 15, 14, 3 and 12 were aberrant in both direct preparations and in those from passaged cells, suggesting that these chromosomes carry genes which, when altered, are particularly important in the multistep process of neoplastic transformation. Most of these chromosomes, or their homologs in other species, have been found to be involved frequently in several different cancers of mice and men, as for example the region on the mouse 15 carrying the Myc and Pvt-1 genes.(ABSTRACT TRUNCATED AT 250 WORDS)