Endoparasitism in captive wild-caught snakes indigenous to Kerala, India.

In this preliminary survey, coprologic examination of six different captive snake species housed in three different herpetariums of Kerala state was conducted from July 2005 to January 2006, to study the prevalence of endoparasites. Of the 25 snakes examined, 22 (88%) were found infected. On the basis of morphology and morphometry, Capillaria spp. (72%), strongyle (28%), spirurid (20%), Strongyloides/Rhabdias spp. (16%), ascarid (12%), pseudophyllidean tapeworm (8%), acanthocephalan (4%) and pentastomid (12%) ova were identified. The occurrence of ova of Hymenolepis spp. as pseudoparasite was also noticed.

Effect of surface area to weight ratio of egg masses on the hatchability of Boophilus microplus eggs.

BACKGROUND & OBJECTIVES: On global basis, ticks transmit a number of pathogens than any other arthropod vector, and are among the most important vectors of diseases affecting humans, livestock and companion animals. Control of the vector has been focused on integrated management involving strategic use of insecticides, use of vaccines, use of herbal acaricides and breed resistance. It has been established that tick vaccine is working on limiting the egg laying potentiality and subsequent hatchability of the ticks fed on immunized animals. To generate entomological data following immunization of animals against ticks an experiment was conducted to establish the role of water content in egg masses for successful hatching into larvae. METHODS: Different size and shape of egg masses of Boophilus microplus, Izatnagar isolate were obtained by manipulating the egg laying process. The weight of the egg masses was measured, keeping their integrity and surface areas of respective egg masses were calculated with the aid of computer software. Larvae hatched from the respective egg masses were counted individually. RESULTS: It is clear that, with an increase in the exposed surface area of the cylindrical egg mass per unit weight, there is a reduction in the number of larvae hatched out. Also, the spherical egg masses significantly (0.026 at 95% confidence level) yielded more larvae per unit weight in comparison to the cylindrical egg masses. INTERPRETATION & CONCLUSION: It has been established that the larval count yielded from an egg mass is more or less dependent on the surface area : weight ratio of the respective egg masses rather than on either the surface area alone or weight of the egg mass alone.

Dihydrofolate reductase as a target for chemotherapy in parasites.

Opportunistic infections are known to cause morbidity and mortality in immunocompromised individuals. In addition, serious infections due to several parasites are also known to affect the quality and duration of life in normal individuals. The importance of dihydrofolate reductase (DHFR) in parasitic chemotherapy arises from its function in DNA biosynthesis and cell replication. DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), an essential cofactor in the biosynthesis of thymidylate monophosphate (dTMP). Inhibition of DHFR leads to a deficiency of dTMP since DHF cannot be recycled, and thus causes inhibition of cell growth. Methotrexate (MTX) and aminopterin (AMT) were among the first known classical inhibitors of DHFR. Trimethoprim (TMP) and pyrimethamine (PYR) are among the first known non classical inhibitors of DHFR. TMP and PYR are selective but weak inhibitors of DHFR from several parasitic organisms and coadministration of sulfonamides is required to provide synergistic effects for clinical utility. Unfortunately, the side effects associated with sulfa drugs in this combination often result in cessation of therapy. Trimetrexate (TMQ) and piritrexim (PTX) are two potent non classical inhibitors, neither of which exhibit selectivity for pathogen DHFR and must be used with host rescue. However, the current combination therapy suffers from high cost, in addition, several mutations have been reported in the active site of parasitic DHFR rendering the infections refractive to known DHFR inhibitors. The selectivity of TMP is a hallmark in the development of DHFR inhibitors and several efforts have been made to combine the potency of PTX and TMQ with the selectivity of TMP. Thus the structural requirements for DHFR inhibition are of critical importance in the design of antifolates for parasitic chemotherapy. Structural requirements for inhibition have been studied extensively and novel agents that exploit the differences in the active site of human and parasitic DHFR have been proposed. This review discusses the synthesis and structural requirements for selective DHFR inhibition and their relevance to parasitic chemotherapy, since 1995.

Heparan sulphate requirement in platelet-derived growth factor B-mediated pericyte recruitment.

HS (heparan sulphate) plays a key role in angiogenesis, by interacting with growth factors required in the process. It has been proposed that HS controls the diffusion, and thus the availability, of platelet-derived growth factor B that is needed for pericyte recruitment around newly formed capillaries. The present paper summarizes our studies on the importance of HS structure in this regulatory process.

Ingress of Bhavnagar City (India).

A preliminary survey of the coastal city-Bhavnagar was undertaken to assess salinity ingress probed through groundwater quality. Water samples from the wells and bores located in the study area were collected and analyzed. Bhavnagar City is found significantly affected by the seawater intrusion. The ground water showed very high values of SO4(-2), Cl(-1), PO4(-3) and, Na(+1), K(+1) compared to the permissible limits for drinking purposes. The quality of ground water in some of the areas was found highly saline and can not be used even for irrigation purpose. The results also indicated a gradual encroachment of seawater into the native ground water.

Correlation of fine needle aspiration cytology, smear and culture in tuberculous lymphadenitis: a prospective study.

BACKGROUND AND AIM: Bacteriological studies are necessary to confirm the diagnosis of tuberculous lymphadenitis, as cytological appearances mimic other granulomatous lesions. The objective was to assess the diagnostic role of culture of fine needle aspiration done on clinically suspected cases of tuberculous lymphadenitis and to determine the prevalence of drug resistance in M. tuberculosis isolates. SETTING AND DESIGN: A prospective, double-blind study over a period of one year in a tertiary care hospital. MATERIAL AND METHODS: Fine needle aspiration cytology and culture were done on 250 patients with clinical suspicion of tuberculous lymphadenitis. STATISTICAL ANALYSIS: Data was statistically analysed using chi square test. Sensitivity, specificity, positive predictive and negative predictive values and likelihood ratio were also calculated. RESULT: Of the 161 cytologically or microbiologically proven cases of tuberculous lymphadenitis, cytological changes consistent with tuberculosis were observed in 133 patients, out of which mycobacteria were isolated in 102 aspirates. Mycobacteria were also isolated from 28 aspirates cytologically missed as tuberculous lymphadenitis. Of the 130-mycobacterial isolates, 5 were non-tuberculous mycobacteria. Culture positivity was significantly higher (P<0.001) than smear positivity. Drug susceptibility studies showed resistance to one or more drugs in 61% of isolated strains with maximum resistance to isoniazid (16% primary and 48% secondary) and minimum to ethambutol (4% primary and 12% secondary). CONCLUSION: Culture for mycobacteria should be carried out on all aspirates from patients suspected with tuberculous lymphadenitis.

Selective cytotoxicity of MIA Pa Ca-2 conditioned medium to acinar cells: a novel approach to reduce acinar cell contaminants in isolated islet preparations from BALB/c mice.

Contamination of acinar cells in islet preparations has been shown to affect islet viability, functionality and yield adversely. Therefore, a strategy which would reduce acinar contamination in islet preparations is much sought. We here demonstrate selective cytotoxicity of conditioned medium (CM) of MIA Pa Ca-2 (human pancreatic carcinoma) cells to acinar cells and suitability of this approach as a simple method of obtaining a pure islet population without affecting their viability and yield. When isolated, islet preparations from BALB/c mice were exposed to conditioned media of MIA Pa Ca-2, acinar cells underwent extensive degeneration to yield 80-85% pure islet population, and islets showed comparable viability to controls not exposed to conditioned media. They also maintained their normal morphology, as assessed by digital image analysis. Islets treated with a conditioned medium also preserved in vitro insulin secretion. Flow cytometric analysis of acinar cells treated with conditioned media revealed accelerated DNA damage (45%), compared to controls (22%). Results emphasize the role of factors secreted by MIA Pa Ca-2 cells in inducing selective toxicity to acinar cells.

Functional knockout of the corepressor CtBP by the second exon of adenovirus E1a relieves repression of transcription.

The C-terminal binding protein (CtBP) acts as a transcriptional corepressor upon recruitment to transcriptional regulators. In contrast, interaction between CtBP and the adenovirus E1A protein is required for efficient activation of E1A-responsive genes, suggesting that E1A might block CtBP-mediated repression. Recruitment of CtBP to a promoter, either as a Gal4CtBP fusion or through an interaction with a Gal4 fusion protein expressing the CtBP interacting domain (CID) of E1A, resulted in transcriptional repression. The second exon of E1A, containing the CID, alleviated repression by Gal4E1ACID-recruited CtBP, but not Gal4CtBP-mediated repression, suggesting that E1A prevented repression by blocking promoter recruitment of CtBP. E1ACID was also sufficient to derepress transcription from several cotransfected promoter constructs. Furthermore, inducible expression of E1ACID in established cell lines resulted in significant changes of endogenous gene expression, possibly by sequestration of CtBP. Together, these data indicated that CtBP might act as a wide-range regulator of transcription. Although CtBP was shown to interact with histone deacetylases (HDACs), transcriptional repression by a Gal4CtBP fusion protein was not sensitive to inhibition of HDACs by trichostatin A (TSA). In contrast, TSA eliminated E1ACID derepression of E1A second exon-responsive promoters. Although the reason for this difference remains to be experimentally verified, it is possible that the requirement for HDACs might differ depending on the mechanism by which CtBP becomes promoter recruited.

Combined effect of nicotinamide and streptozotocin on diabetic status in partially pancreatectomized adult BALB/c mice.

Pancreatic regeneration after pancreatectomy has been well documented in the animal models. We have recently reported that STZ diabetic animals operated for partial pancreatectomy showed normoglycemic status after the operation as compared to uncontrolled hyperglycemia and even death in the diabetic sham operated animals. In drug and virus-induced experimental diabetic models there is a high mortality of animals due to uncontrolled destruction of the beta-cells. In order to destroy sufficient beta-cell mass so as to induce diabetes but prevent mortality, we designed present studies to investigate the combined effect of pancreatectomy, nicotinamide, and streptozotocin (STZ) on diabetic status of BALB/c mice. BALB/c mice of either sex were subjected to 50% pancreatectomy. These were then treated with nicotinamide (350 mg/kg body weight) before and after streptozotocin (200 mg/kg body weight) administration. The changes in body weight, blood glucose levels, serum and pancreatic insulin contents of these animals were monitored in experimental and control group for 12 weeks, and follow up studies were made of these animals for further 12 weeks. It was found that there was a drastic loss of body weight, decreased serum and pancreatic insulin levels coupled with sustained and low levels of hyperglycemia in the experimental group as opposed to the control group. The results indicate that partial pancreatectomy followed by nicotinamide and streptozotocin treatment leads to a long-lasting hyperglycemic state, depicting the clinical symptom of NIDDM without mortality. The study probably reveals a new model for experimental diabetes.

The Arabidopsis COMATOSE locus regulates germination potential.

Mutation of the COMATOSE locus in Arabidopsis results in a marked reduction in germination potential. Whilst the morphology of comatose (cts) embryos is not altered, physiological analysis reveals that mature cts seeds do not respond to gibberellin. Prolonged chilling of imbibed seeds only partially restores germination potential, and seeds do not after ripen. Genetic analysis shows that the cts phenotype is expressed in the embryo and phenotypic differences between wild-type and mutant plants were not observed during other stages of plant growth and development. Therefore cts represents a new class of mutant, with a specific lesion that results in severely impaired germination potential. Genetic interactions were analysed between cts and loci that regulate embryo maturation, and abscisic acid biosynthesis and perception. Results from these studies showed that the cts mutant phenotype required the wild-type action of these loci, and suggested that CTS exerts a repressive function on these loci. A model is presented postulating that CTS promotes increased germination potential, and represses embryo dormancy. These functions of CTS may result in the removal of embryo dormancy as a prerequisite to germination.

Bio-artificial liver from cultured human foetal hepatocytes: feasibility and prospects.

BACKGROUND: Conventional therapy for acute liver failure has not been able to improve survival beyond 40%. Apart from liver transplantation, the most promising development in this field is the utilization of cultured hepatocytes to make 'bio-artificial liver support systems' as a 'bridge to transplantation' or ideally as a 'bridge to total recovery'. This study examines the feasibility of culturing foetal hepatocytes without the use of growth factors and formulating a bio-artificial liver support device in our set-up. METHODS: Foetal hepatocytes were harvested from the liver obtained from mid trimester abortions at Armed Forces Medical College and Command Hospital (SC), Pune. The liver was perfused with Phosphate Buffered Saline (PBS) and collagenase type IV and was cut with a pair of sterile scissors into tiny pieces. Cells so separated, were washed with PBS plus foetal calf serum and stirred to disperse the cell aggregates. Filtered cell suspensions were inoculated in polystyrene flasks containing hepatocyte culture medium (MEM E: 75%, M199: 25%, BSA: 0.1%, Bovine Insulin 5 micrograms/ml, FCS: 10%, Penicillin: 10 i.u., Streptomycin 50 micrograms/ml, Hydrocortisone 5 micrograms/ml and incubated at 37 degrees C. The functional capabilities of the cultured hepatocytes were analyzed by studying production of albumin and a foetoprotein. Structural integrity of hepatocytes was assessed by light and electron microscopy. RESULTS: The hepatocyte yield varied from 2 to 60 x 10(6) cells/L with an average of 38 x 10(6) cells/L in the eight consecutive experiments. Initial hepatocyte viability varied from 25% to 90% with an average of 61%. The yield and the viability of hepatocytes were adversely affected by the condition of foetus at birth and use of intra-amniotic injections for inducing abortions. Hepatocyte monolayers and colonies formed in 75% experiments. The cultures could be maintained in incubation without the use of epidermal or hepatocyte growth factors for 2-25 days with a mean survival of 8.9 days. The cells in culture were found to be structurally normal and functionally active and could be cryo-preserved. These hepatocytes were inoculated into a hollow fiber module to formulate bio-artificial liver support device. The cultures ultimately developed either cellular disintegration or bacterial infections despite use of antibiotics in the culture medium. CONCLUSIONS: We conclude that it is feasible to maintain foetal hepatocyte cultures without the use of expensive growth factors for over 8 days. Bio-artificial liver formulated with cultured foetal hepatocytes is now a step closer to clinical trials.

Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1.

The Avena fatua (wild oat) homologue of VIVIPAROUS 1 (AfVP1) has been implicated in controlling the maintenance of embryo dormancy in mature imbibed seeds, but the detailed mechanisms by which this transcription factor family activates embryo maturation pathways and simultaneously represses germination are not known. A two-hybrid screen in yeast identified three proteins that interacted specifically with AfVP1 (AfVP1 interacting proteins; AfVIPs). AfVIPs 2 and 3 interacted with the C-terminus of AfVP1, which contains the B2 + B3 domains, previously shown to bind DNA, whereas AfVIP1 interacted with the isolated B3 domain. Using purified proteins in in vitro experiments, all three AfVIPs were shown also to interact with the Arabidopsis homologue ABSCISIC ACID INSENSITIVE 3 (ABI3). The three AfVIPs were expressed in both dormant and non-dormant embryos, but the abundance of AfVIP1 and 3 transcripts was greater in germinated than dormant seeds, whereas transcripts of AfVIP2 (and AfVP1) were more highly expressed in dormant embryos. The AfVIP3 protein has homology to a human cell-crisis gene with a predicted role in the cell cycle; AfVIP2 contains a ring-type zinc finger motif. These homologies, together with analysis of expression studies, suggest that these proteins may play specific roles in AfVP1-mediated regulation of the dormancy to germination transition in A. fatua seeds.

Interactions of the developmental regulator ABI3 with proteins identified from developing Arabidopsis seeds.

The ABI3 locus is a major regulator of embryo development in Arabidopsis and is essential for the simultaneous activation of the maturation pathway, as well as repression of germination and seedling development. We used a two-hybrid screen in yeast in order to identify proteins that interact with ABI3. Four ABI3-interacting proteins (AIPs) were identified which showed specific in vivo and in vitro interactions with the C-terminal region of ABI3 that contains the B2 and B3 domains, previously shown to have DNA binding activity. The expression characteristics of the genes encoding the AIPs have also been analysed in wild-type and abi3, lec1 and fus3 embryo mutants. This analysis demonstrated differential expression of these genes during normal embryo development and in the mutant lines. All the AIPs show homology to existing transcription factors and therefore they may function with ABI3 within the network of transcriptional regulators that control embryo development in Arabidopsis.

Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations.

The S3 allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S1 allele, preceded by an 18 amino acid signal sequence. Expression of the S3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S1 protein. In contrast to other S proteins identified to date, S3 protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of Western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S3 allele with antiserum raised to S3e, revealed a protein (S3s) which was indistinguishable in pI and Mr from that in the Shirley population. A cDNA encoding S3s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.