Molecular evidence of hepatozoonosis in tigers of Vidarbha region of Maharashtra State of India.

BACKGROUND: Hepatozoonosis has been reported in many species around the world. Few incidences have been reported in various species of wild felids. Tigers are endangered large cats and are protected under the Wildlife Protection Act, 1972 under Schedule I. The study was carried out to estimate the positivity rate of hepatozoonosis in tigers of the Vidarbha region of Maharashtra, India. METHODS: Blood (n = 21) or tissue samples (n = 5) were collected from 26 wild captured / zoo-born or dead tigers during the quarantine period/post-mortem examination. Blood smear examination along with Polymerase Chain Reaction (PCR) studies were conducted for the detection of hepatozoonosis. All the amplicons from the positive samples were purified and sequenced, and the sequences were subjected to nBLAST analysis to detect the species of Hepatozoon. The sequences were deposited into public domain database of National Center for Biotechnology Information (NCBI) and accession numbers were allotted. A phylogenetic study was undertaken to understand the evolutionary lineage of the pathogen. Tissue distribution studies were carried out on tissue samples received during post mortem. A clinical case in a tiger cub was managed and sub-clinical cases were monitored for relapse. Age-wise, sex-wise, region-wise and captive time-wise positivity rate was estimated. The data was analyzed using statistical tools. RESULTS: A total of 12 tigers were found positive for H. felis during the screening. A clinical case was diagnosed and successfully treated. The age group of 0-3 years reported a positivity rate of 66.66%, and all the cases found positive were reported between the age group of 0-7 years. Males reported a positivity rate of 58.33 per cent, while females reported 35.71%. Taboba and Andhari Tiger Reserve of the state had a positivity rate of 52.94 per cent. However, the statistical analysis for blood parameters and positivity rate by 't' test and Chi-squared test were found to be non-significant. CONCLUSIONS: An overall positivity rate of 46.15% indicates the wide distribution of hepatozoonosis among wild tigers of the Vidarbha region of Maharashtra, India, which is strategically important considering the gene flow and migration of tigers. Hepatozoonosis can progress to clinical outcomes in young animals and require veterinary intervention. Molecular tools and phylogenetic studies can supplement important data on circulating species of Hepatozoon in the field. Further studies on the clinical management and epidemiology of the infection in wild felids will comprehend the cause of wildlife conservation.

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans.

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.

Effect of mild whole body hyperthermia on cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine in Swiss mice.

Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a modifier of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) cytotoxicity in mice. Adult Swiss male mice, 6-8 weeks old, weighing about 25 g were exposed to mild WBH (39 degrees C, 1 h) in a precision temperature controlled environmental chamber maintained at 50-60% relative humidity. Twenty-four hours after treatment, animals were administered with different doses of MNNG either by intraperitoneal (i.p.) injections or by feeding through drinking water and were monitored for survival. The studies revealed that the exposure of animals to mild WBH, 24 h prior to MNNG administration results in an increase in survival and recovery in mean body weight compared with those administered with MNNG only. This suggests that prior WBH treatment can effectively reduce the MNNG cytotoxicity in mice.

Radioprotective effect of whole-body hyperthermia on mice exposed to lethal doses of total-body gamma irradiation.

The whole-body hyperthermia (40 degrees C, 1 hr) 20-48 hr prior to total-body irradiation (TBI) with 9 Gy gamma rays gave 80% protection as assessed by survival of the animals. However this was reduced to 50% when mice were irradiated 7 or 15 days after hyperthermia. The local hyperthermia (42 degrees C, 1 hr) given prior to irradiation, on the other hand, did not show any protective effect. The whole-body or local hyperthermia given after TBI had no protective effect on survival of animals.

A sensitive assay for Staphylococcus aureus nucleases.

A sensitive assay for staphylococcal nuclease involving incubation of the enzyme sample with heat-denatured [3H]thymidine labelled DNA from E. coli, precipitation with trichloroacetic acid and measurement of the radioactivity of acid-soluble nucleotides released has been developed. The assay is sensitive enough to be used for comparing the levels of nucleases elaborated by different strains of S. aureus as well as for determining the extent of contamination of S. aureus in food and water samples even at levels at which the conventional spectrophotometric and toluidine blue-DNA methods are totally inadequate.

Swine flu (H1N1 influenza): awareness profile of visitors of swine flu screening booths in Belgaum city, Karnataka.

The 2009 flu pandemic was a global outbreak of a new strain of H1N1 influenza virus often referred colloquially as "swine flu". The objectives of the study were: (1) To know the sociodemographic and awareness profile of visitors attending swine flu screening booths. (2) To reveal sources of information. The present cross-sectional study was undertaken among the visitors (18 years and above) attending swine flu screening booths organised within the Belgaum city during Ganesh festival from 28-08-2009 to 03-09-2009 by interviewing them using predesigned, pretested structured questionnaire on swine flu. The data was collected and analysed using SPSS software programme for windows (version 16). Chi-square test was applied. Out of 206 visitors, 132 (64.1%) were males and 107 (51.9%) were in the age group of 30-49 years; 183 (88.8%) had heard about swine flu. More than a third of the visitors (38.3%) disclosed that there was a vaccine to prevent swine flu. Majority responded that it could be transmitted by being in close proximity to pigs (49.0%) and by eating pork (51.5%). Newspaper/magazine (64.6%), television (61.7%), and public posters/pamphlets (44.2%) were common sources of information. The present study revealed that doctors/public health workers have played little role in creating awareness in the community. The improved communication between doctors and the community would help to spread correct information about the disease and the role that the community can play in controlling the spread of the disease.

Syntheses of DNA and chromosomal proteins in the rat thymus following whole-body X-irradiation.

Syntheses of chromosomal proteins was studied in relation to DNA syntheses in the rat thymus following whole-body X-irradiation (600 rad). There was a considerable depression of the syntheses of DNA and histones during the first 4--48 hours after irradiation. The syntheses of histones H3 and H4 plus H2A were affected to a much greater degree than those of histones H1 and H2B, suggesting a tight coupling between the syntheses of DNA and histones H3 and H4 plus H2A. A part of the lysine-rich histones H1 and H2B, however, seems to be synthesized, even in the absence of DNA synthesis. Biosynthesis of non-histone proteins was also depressed in the thymus after irradiation. The degree of inhibition, however, was very low, except for the syntheses of a few non-histone protein components which were depressed to a considerable extent. This implies that the synthesis of the majority of non-histone proteins is independent of DNA synthesis.

Development of rural girl children -- a bias.

PIP: In Dharwad taluk, Dharwad District, Karnataka, India, researchers followed the development and growth of 274 infants from birth to 5 years. The proportion of well-nourished girls decreased as the age increased for both sexes. The proportion of well-nourished girls was lower than boys up to age 15 months. Between 15 months and 42 months, it was higher. In all degrees of malnutrition, the proportion of females was higher than the males. In terms of motor and mental indices, at 27 months, no girl was well-nourished. Between birth to 8 months, the motor development indices of the well-nourished girls was slightly higher than that of boys. The motor indices were equal for both sexes between 9 and 11 months. At 12, 18, and 24 months, well-nourished girls had higher motor indices with significant difference at 24 months (p 0.001). The disparity in motor indices was greater for malnourished girls than malnourished boys, which was significant between 9 and 12 months (p 0.01-0.05). In other words, the motor capacity of malnourished infant girls was much better than their male counterparts. Between birth and 8 months, mental development indices (MDIs) of the well-nourished girls were higher than boys with the difference being significant at 2 months (p 0.05). Thereafter, they were higher for boys than girls with the differences being significant at 24 and 30 months (p 0.01). Among malnourished children, girls had higher MDIs than boys with significant differences at 9 and 21 months (p 0.01). MDIs decreased as age increased. They show that, in absence of parental encouragement, girls did better in mental development than boys despite being malnourished. These findings suggest that girls are neglected at birth in terms of nutritional status and performance in mental and motor tasks. Thus, parents need to be educated to avoid depriving girls of nutrition and mental and motor stimulation.^ieng

Effect of whole-body irradiation on transcription in the rat thymus.

Studies were carried out to elucidate the mechanism underlying the impairment in RNA synthesis in the rat thymus after whole-body exposure to X-rays (1000 rad). The ability of thymus nuclei to catalyse RNA polymerization in vitro was adversely affected in irradiated rats. Further experiments showed that whole-body irradiation caused a considerable decrease in both the activity of RNA polymerase and the template efficiency of chromatin but not in that of DNA isolated from chromatin. These radiation-induced changes in transcription seem to arise partly from the direct effect of radiation on the thymus itself and partly from the abscopal mechanism(s).

Radiation-induced thymine base damage and its excision repair in active and inactive chromatin of HeLa cells.

The extent of production and excision repair of 5,6-dihydroxydihydrothymine type base (t') damage was determined in transcriptionally active and inactive chromatin of HeLa cells after exposure to 6.8 MeV electrons. It was observed that not only the yield but also rate of repair of t' products was greater in the active chromatin compared to the inactive chromatin of HeLa cells. The results strongly indicate that the conformation of chromatin is an important factor in determining the sensitivity to radiation damage and accessibility to enzymes required for repair of such damage.

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function.

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3-4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70-85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20-25% of the nuclear DNA polymerase alpha was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replication via-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. II. Effect of ATP on free and matrix bound TdT.

Endogenous nuclease digestion of thymus nuclei from 3-4 week old rats followed by a step wise extraction with low salt, 0.5 M salt and 1 M salt removed approximately 70-85% of total nuclear terminal deoxynucleotidyl transferase (TdT) whereas approximately 15-30% of the enzyme remained tightly bound to the residual nuclear matrix. The cytoplasmic TdT as well as the bulk of nuclear TdT extracted in low salt and 0.5 M salt was found to be strongly inhibited at low concentration of ATP whereas matrix bound TdT and a significant portion of the enzyme in 1 M salt extract was completely insensitive to this nucleotide. The ATP resistant enzyme in the 1 M salt extract was unstable and slowly converted to ATP sensitive form upon prolonged preincubation on ice whereas under similar conditions it remained unaffected in the matrix bound form. These observations lead us to suggest that ATP resistant matrix bound TdT being capable of discriminating unnatural rNTPs against the natural dNTP substrates, may be the functionally organized form of the enzyme and that free TdT having lost the capability to distinguish between dNTP and rNTP may be the nonfunctional form of the enzyme in the thymus gland.

Characterisation of two intronic nuclear-matrix-attachment regions in the human DNA topoisomerase I gene.

We identify two high-affinity matrix-attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements, designated MI and MII, are specifically bound by the nuclear matrix and partition with scaffolds in vitro. One of these MAR elements, MII, is part of a genomic region which is hypersensitive for endogenous nucleases. We have sequenced both DNA elements and have characterized their mode of binding to the nuclear matrix. Experiments with the minor-groove-binding ligands distamycin and chromomycin indicate that the A+T-rich regions, most likely homopolymeric A tracts, are responsible for binding of these DNA elements to the nuclear matrix. MII contains an alu-like element and a segment of curved DNA. Analysis of subfragments of MII show that the curved DNA region itself shows only weak nuclear-matrix binding, and that the high-affinity binding sites are located on subfragments on the 5' side of the curved DNA. In addition, we found that the alu-like sequence does not contribute significantly to the binding of MII and of subfragments of MII to nuclear-matrix proteins. Comparing the distribution of repetitive sequences in the cloned parts of human DNA topoisomerase I gene with the location of high-affinity matrix-binding sites we find no evidence that repetitive DNA may be located close to MAR elements as has been previously suggested.

DNA synthesis in nuclei and nuclear matrices of regenerating rat liver: effect of whole-body gamma irradiation.

Partial hepatectomy (PH) of rats (Wistar strain) resulted in acceleration of DNA synthesis in liver which reached a maximum at 36 h after PH. Whole-body radiation exposure (10 Gy) of the rats at 12 h after PH completely arrested this stimulation in DNA synthesis. The elevation of DNA synthetic rate in response to PH and complete obliteration of this stimulation by whole-body radiation exposure were found to be the reflection of levels of DNA polymerase-alpha in nuclei and nuclear matrices isolated from the rat livers. Studies based on assays of DNA polymerase in nuclei and nuclear matrices, with and without exogenous DNA template (activated calf thymus DNA), revealed that whole-body irradiation blocked induction of DNA polymerase-alpha and, in turn, assembling of DNA polymerizing apparatus. Irradiation of nuclei (suspended in buffer) in vitro at doses as high as 500 Gy did not have any inhibitory effect on DNA polymerase-alpha activity.

Site directed mutagenesis of DNA polymerase I (Klenow) from Escherichia coli. The significance of Arg682 in catalysis.

We have reported that a domain containing Arg682 in the Klenow fragment of Escherichia coli DNA polymerase I (pol I) is important for the template-dependent dNTP-binding function [Pandey, V.N., Kaushik, N. A., Pradhan, D. S. & Modak, M. J. (1990) J. Biol. Chem. 265, 3679-3884]. In order to further define the role of Arg682 in the catalytic process, we have performed site-directed mutagenesis of this residue. For this purpose the Klenow-coding region of the DNA-pol-I gene was selectively amplified from the genomic DNA of E. coli and was cloned in an expression vector, pET-3a. This clone under appropriate conditions overproduces the Klenow fragment in E. coli. Using this clone (pET-3a-K) as the template, two mutant polymerase clones were constructed in which arginine has been replaced with either alanine, [R682A] pol I, or lysine [R682K] pol I. Both mutant enzymes showed significantly lower specific activity as compared to the wild-type enzyme. The kinetic analyses of the mutant enzymes indicated a 3-4-fold increase in the Km for the substrate dNTP, a 20-25-fold decrease in the Vmax and an overall decrease in the processive nature of DNA synthesis in both the mutant enzymes. The reverse mutation of Ala682 to the wild-type form Arg682 fully restored the processive nature and the polymerase activity of the enzyme. These observations suggest that the positively charged guanidino group in the side chain of Arg682 is catalytically important but not absolutely essential for synthesis of DNA. Furthermore it appears to maintain high processivity of the DNA synthesis catalyzed by the enzyme.

Terminal deoxynucleotidyltransferase containing megadalton complex from young rat thymus nuclei: identification and characterization.

Nuclear matrix prepared from 2-3 week old rat thymuses contains tightly bound TdT activity which has been quantitatively solubilized with nonionic detergent and sonication. TdT is contained in a discrete complex with a sedimentation value of 23 S. The complex is retained on an anti-TdT antibody column and contains DNA ligase and 3'-5' exonuclease activities as well as DNA and several other proteins but is devoid of replicative DNA polymerases. Such a type of multienzyme complex is absent from the nuclear extracts of thymus prepared from older rats and also from liver and spleen extracts of young and old rats.

Effect of whole body hyperthermia on radiation therapy of transplanted fibrosarcoma in Swiss mice.

The exposure of normal mice to whole body hyperthermia (1 h WBH at 39 or 40 degrees C), 20 or 48 h prior to total body irradiation (TBI) with lethal doses of gamma-rays affords significant protection as assessed by survival. The radioprotective effect of WBH, as observed in normal mice, diminished in tumour bearing mice depending upon the size of tumour. Treatment of tumour bearing mice with mild WBH, 20 h prior to local irrradiation (LIR), did not protect the transplanted tumour against radiotherapy with a single dose of 20 Gy or fractionated dose (in five fractions) of 7.5 Gy on alternate days. In fact, mild WBH treatment enhanced the tumour regression and increased the mean survival time after fractionated dose therapy. However, the prior mild WBH was found to be ineffective in protecting normal tissue, as assessed by skin contraction after local irradiation (50 Gy). This indicates that mild WBH treatment given 20 h prior to local radiotherapy enhances fibrosarcoma tumour regression but cannot protect skin (normal tissue) against local irradiation. It appears that radioprotection of animals by WBH may be the consequence of its radioprotective effect on haemopoietic tissues mediated through certain cytokines. Perhaps WBH may not have a radioprotective effect on other tissues, as evident from skin contraction studies.