Bacillus thuringiensis strains from Western Ghats of India possess nematocidal property against Haemonchus contortus larvae of goats.

Nematocidal properties of spore crystal mixtures of six Bacillus thuringiensis (Bt) strains (KAU 49, 50, 52, 61, 99 and 424) collected from Western Ghats, a biodiversity hot spot of India, were analysed against Haemonchus contortus larvae isolated from goats. One dose nematocidal assay dose response to lyophilised spore-crystal mixtures (SCM) of the six Bt strains were determined by adding 200 µg/mL of each SCMs to culture plate wells containing aqueous suspension of H. contortus larvae. Out of the strains screened, KAU 50 and 424 were found to possess nematocidal properties. Maximum nematocidal properties were exhibited 7 days post-inoculation of the lyophilised SCMs. The 50 per cent lethal concentrations deduced by log probit analysis for KAU 50 was found to be 130.59 µg/mL, whereas that of KAU 424 was found to be 144.536 µg/mL at 95 per cent confidence level. This is the first report on the nematocidal propery of Bt strains against Haemonchus contortus larvae isolated from goats. Further studies are needed for identification and characterisation of the toxin.

Crystal Protein of a Novel Bacillus thuringiensis Strain Inducing Cell Cycle Arrest and Apoptotic Cell Death in Human Leukemic Cells.

Parasporal inclusions of a native non haemolytic Bacillus thuringiensis strain KAU 59 was screened for its cytotoxicity against human lymphocytic leukemic cell line jurkat and normal human lymphocytes. The cytotoxicity of proteinase activated and non activated solubilised parasporal inclusions against both cell lines was assessed by Cell Titer 96 Aqueous Non Radioactive Cell Proliferation Assay Kit using MTS. The 50 per cent effective concentration (EC50) values were deduced from log probit analysis at 48 h. Morphological changes associated with cytotoxicity were evaluated and molecular mechanisms of cell death were elucidated by TUNEL assay at 48 h post-inoculation. The fluorescence assisted cell sorting was done in the flow cytometer to assess the stage of cell cycle arrest. Relative quantification of caspase-3 expression in Jurkat cells treated with parasporal inclusion protein of KAU 59 was done by qRTPCR The results indicated that the protein was cytotoxic to jurkat cells at the same time non toxic to normal lymphocytes. Cytotoxicity was evident only after proteolytic activation. Apoptotic cell death was confirmed in the protein treated cells by TUNEL Assay and also up regulated caspase-3 gene expression (P < 0.001). S phase cell cycle arrest was confirmed by and fluorescence associated cell sorting.

Preparation and evaluation of biocomposites as wound dressing material.

Collagen was isolated from the chrome containing leather waste (CCLW) which is a major solid waste in leather industry. Composite films were made using sago starch (SG), soya protein (SY), and collagen (C) and were cross linked with glutaraldehyde (G).The films prepared were characterized for their physico chemical properties like tensile strength, infrared spectra, thermogravimetric analysis, surface morphology, and water absorption studies. Better mechanical properties and surface morphology were observed for SG-SY-G-C films compared to other films prepared using collagen. The composite films prepared were used as wound dressing material on the experimental wounds of rats and healing pattern was evaluated using planimetric, biochemical, and histopathological studies. These studies have revealed better wound healing capacity of SG-SY-G-C film and utilization of CCLW in the preparation of value added product like wound dressing material.

Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice.

BACKGROUND & AIMS: Post-mitotic cells such as brain and heart cells are particularly vulnerable to oxidative damages during ageing. In this study, we evaluated the effect of Ganoderma lucidum on the antioxidant status in the mitochondria of heart and brain of aged mice. METHODS: The effect was evaluated by estimating the activities of manganese-superoxide dismutase (Mn SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and catalase (CAT) as well as levels of reduced glutathione (GSH), lipid peroxidation, advanced oxidation protein products (AOPP) and reactive oxygen species (ROS) in the heart and brain mitochondria of aged mice after oral administration of ethanolic extract of G. lucidum (50 and 250mg/kg), once daily for 15 days. The effect was compared with that of aged and young control animals. dl-alpha-lipoic acid (100mg/kg) was taken as the positive control. RESULTS: Administration of G. lucidum extract significantly (p<0.05) elevated the levels of GSH as well as activities of Mn SOD, GPx, and GST and decreased significantly (p<0.05) the levels of lipid peroxidation, AOPP and ROS. CONCLUSION: G. lucidum administration could improve the age-related decline of antioxidant status which was partly ascribed to free radical scavenging activity.

Regulation of Caspase-3 and Bcl-2 Expression in Dalton's Lymphoma Ascites Cells by Abrin.

The role of abrin, a toxic lectin isolated from seeds of Abrus precatorius Linn in inducing apoptosis in murine Dalton's Lymphoma Ascites (DLA) cells was evaluated. Abrin when incubated at the concentration of 10 ng per million DLA cells could bring about cell death as typical morphological changes with apoptosis. However, necrotic cell death dominated when a higher dose of abrin was used. DNA samples, isolated from DLA cells treated with abrin showed fragmentation. Abrin brought about induction of apoptosis by stimulating the expression of pro-apoptotic Caspase-3, at the same time blocking the expression of Bcl-2, which is an anti apoptotic gene. However, the expression of tumor suppressor gene p53 has not been observed in control and abrin-treated DLA cells. Results suggested that abrin effectively induced apoptotic changes in the tumor cells that led to cellular death.

Brahma Rasayana enhances in vivo antioxidant status in cold-stressed chickens (Gallus gallus domesticus).

OBJECTIVE: To evaluate the antioxidant status of chicken during cold stress and to investigate if there are any beneficial effects of Brahma Rasayana supplementation in cold stressed chicken. MATERIALS AND METHODS: Activities of enzymatic and levels of non-enzymatic antioxidants in blood / serum and liver tissue were evaluated in chicken exposed to cold (4 +/- 10C and relative humidity of 40 +/- 5%, for six consecutive hours daily, for 5 or 10 days). The antioxidant properties of Brahma Rasayana (BR) supplementation (2 g/kg daily, orally) during cold stress was also studied. RESULTS: There was a significant (P < 0.05) decrease in antioxidant enzyme in the blood, such as, superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), and serum reduced glutathione (GSH) in cold stressed chicken. Serum and liver lipid peroxidation levels were significantly (P < 0.05) higher in cold stressed untreated chickens when compared to the treated and unstressed groups. There was also a significant (P < 0.05) increase in the antioxidant enzymes in the blood, such as, catalase (CAT) and SOD, in the liver CAT and SOD, and in GPX and GR in BR-treated cold stressed chicken, when compared to the untreated controls. CONCLUSIONS: Results of the present study conclude that in chicken, BR supplementation during cold stress brings about enhanced actions of the enzymatic and non-enzymatic antioxidants, which nullify the undesired side effects of free radicals generated during cold stress.

Amelioration of heat stress induced disturbances of antioxidant defense system in chicken by brahma rasayana.

Since the range of comfort zone or thermo neutral zone of domestic chickens is narrow, they become easily susceptible to heat and cold environmental stress. We evaluated Brahma Rasayana (BR) supplementation on concentrations of certain oxidative stress markers associated with heat stress. A total of 48 egg type male chickens of local strain were divided into six groups (n = 8) for the study. Three groups were fed with BR orally at the rate of 2 g/kg bw daily for 10 days prior to and during the period of experiment. Two of the four groups that were exposed to heat stress (HST i.e. to a temperature of 40 +/- 1 degrees C and relative humidity of 80 +/- 5% in an environmental chamber) for 4 h daily for 5 or 10 days, received BR orally. The other two groups remained as BR treated and untreated non-heat stressed (NHST) controls. There was a significant (P < 0.05) increase in the activities of antioxidant enzymes in blood such as catalase (CAT) and superoxide dismutase (SOD), as well as liver CAT, glutathione peroxidase (GPX) and glutathione reductase (GR) in NHST-BR treated and HST-BR treated (both 5 and 10 days) chickens when compared with untreated controls. A great deal of significant (P < 0.05) variations were seen in serum and liver reduced glutathione (GSH) concentration in NHST-BR treated and HST-BR treated (both 5 and 10 days) chickens. Serum and liver lipid peroxidation levels were found to be significantly (P < 0.05) higher in HST-untreated (both 5 and 10 days) chickens when compared with other groups. Thus BR supplementation during HST brings about enhanced action of enzymatic and non-enzymatic antioxidants, which nullified the undesired side effects of free radicals that are generated during HST.

Effect of abrin on cell-mediated immune responses in mice.

Effect of abrin isolated from Abrus precatorius on the cellular immune responses was studied in normal as well as tumor-bearing animals. Administration of abrin was found to enhance the proliferation of splenocytes and thymocytes (lymphocytes in general) in responses to mitogens. Natural killer cell activity was enhanced significantly by abrin in both the normal (49.8% cell lysis on day 9) and the tumor-bearing group (51.7% cell lysis on day 9), and it was found to be earlier than the control. Antibody dependent cellular cytotoxicity was enhanced in the abrin treated tumor-bearing group on the ninth day (44% cell lysis). An early antibody dependent complement mediated cytotoxicity was observed in the abrin treated group on day 15 (27.6% cell lysis). Results of our present study suggest the immunomodulatory property of abrin.

Serum profile of calcium, phosphorus and magnesium in crossbred heifers as influenced by gestation and early lactation.

The serum profile of certain macroelements such as calcium, inorganic phosphorus and magnesium of healthy crossbred heifers in Kerala were estimated before and after conception, at different stages of pregnancy and early lactation. The serum concentration of calcium showed a decreasing trend during second trimester of pregnancy which attained significantly lower (P < 0.01) value (5.57 mg/dl) when compared to controls. By the ninth month of pregnancy calcium level was elevated. The serum phosphorus level was significantly higher (P < 0.01) during the fifth month of pregnancy (8.29 mg/dl) when compared to controls and then it was decreased by the 9th month of pregnancy. The serum magnesium level showed an increasing tendency to attain a peak value (2.55 mg/dl) by the 9th month of pregnancy which was significantly higher (P < 0.01) when compared to the control. During the period of study no production diseases were encountered in the animals screened which indicated that the serum calcium, phosphorus and magnesium profile of these animals were at the optimum level.

Antitumour effect of abrin on transplanted tumours in mice.

Abrin, a galactose specific lectin was purified using sepharose 4B affinity column from seeds of Abrus precatorius. It exhibited antitumour activity in mice when used at a sublethal dose of 7.5 micrograms/kg every alternate day for 10 days. Both intralesional and intraperiloneal (i.p.) administration of abrin was effective in reducing solid tumour mass development induced by Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cells. DLA cell line was more sensitive to abrin than EAC. Abrin when injected i.p. increased the life span of ascites tumour bearing mice. Abrin when used simultaneously with tumour cells brought about maximum antitumour effect. On developed tumour masses, abrin administration brought about significant reduction in tumour volume, especially in DLA induced tumours. Prophylactic administration of abrin was found ineffective.

Immunopotentiating activity of abrin, a lectin from Abrus precatorius Linn.

A non-toxic dose of abrin, (1.25 microg/kg body wt) consecutively for five days in normal mice stimulated specific humoral responses. A noticeable increase was observed in total leucocyte count, lymphocytosis, weights of spleen and thymus, circulating antibody titre, antibody forming cells, bone marrow cellularity and alpha-esterase positive bone marrow cells. The results suggest that abrin can potentiate the humoral immune response of the host.

Assessment of physiological stress in periparturient cows and neonatal calves.

Pregnancy is considered to be one of the physiological stressors. The stress hormone, cortisol is significantly involved in various events during periparturient period including initiation of parturition. The study was conducted to estimate the serum cortisol concentration in cows and the neonatal calves in order to correlate the effect of cortisol on certain haematological and biochemical parameters such as blood glucose level (BGL), total plasma protein (TPP), lymphocyte:neutrophil ratio and mitogen induced lymphocyte proliferative response. Blood samples were collected from six cows in four periods, namely., 3 days prior to parturition, on the day of parturition, and 7 days after parturition. Blood samples were also collected from neonatal calves in the period 0, 7 and 14 days of age. Calves above two months of age and non-pregnant dry cows were considered as the controls. The serum cortisol concentration in cows on the day of parturition was significantly higher (P < 0.01) than controls and the value in calves was also significantly higher (P < 0.01) at 0 day than their controls. On the day of parturition BGL level of the dam and calves were significantly higher (P < 0.01), whereas the proliferative response of lymphocytes to mitogen was significantly lower (P < 0.01) than controls. However TPP levels did not differ significantly. This confirmed that the dam at the time of parturition and neonatal calf before taking colostrum are under a high risk of infection because of the low profile of immune status. The lymphocyte:neutrophil ratio also justified the above suggestion.

Expression in transgenic mice of dominant interfering Fas mutations: a model for human autoimmune lymphoproliferative syndrome.

Most humans with autoimmune lymphoproliferative syndrome (ALPS) carry heterozygous dominant mutations in one allele of the gene encoding Fas/APO-1/CD95. ALPS patients, like Fas-deficient MRL lpr/lpr mice, have lymphoproliferation, autoimmunity, increased CD4(-)/CD8(-) T lymphocytes, and apoptosis defects. Consistent with the phenotypic variability of lpr/lpr mice of different background strains, human genetic studies indicate that a Fas mutation is insufficient to induce ALPS in all mutation carriers. To investigate the dominant function of human Fas mutations and the additional genetic factor(s) involved in the development of ALPS, we generated transgenic mice expressing, in addition to endogenous Fas, mouse Fas molecules bearing mutations in the intracellular death domain corresponding to mutations identified in ALPS patients. Transgenic mice developed mild features of ALPS, including hepatosplenomegaly, elevated proportions of lymphocytes in spleen and lymph nodes, apoptotic defects, and hepatic lymphocytic infiltrates. Therefore defective murine Fas proteins act in a dominant manner to impair apoptosis of activated lymphocytes and disrupt lymphocyte homeostasis. The influence of genetic background on phenotype was studied by comparing transgenic mice on FVB/N and (FVB/N x MRL) backgrounds with syngenetic control mice and with MRL and MRL lpr/lpr mice. While expression of transgenic mutant Fas contributed mainly to hepatosplenomegaly and accumulation of lymphocytes, MRL background genes played a major role in the production of autoantibodies and elevated serum immunoglobulin levels. Moreover, compared to FVB/N (+/+) mice, a substantial Fas-specific apoptotic defect was found in MRL (+/+) mice, suggesting a mechanism for the known tendency of this strain to develop autoimmunity.

Specificity analysis of sera from breast cancer patients vaccinated with MUC1-KLH plus QS-21.

The mucin MUC1 is expressed on breast cancers in an underglycosylated form compared to normal tissues and is therefore a potential target for cancer immunotherapy. MUC1 contains multiple tandem repeats of the 20 amino acid (aa) peptide (VTSAPDTRPAPGSTAPPAHG). The APDTRPA epitope is particularly immunogenic since it is recognized by a variety of murine monoclonal antibodies and by some sera and cytotoxic T-cells from unimmunized patients with epithelial cancers. We have prepared a 30 aa peptide (C)VTSAPDTRPAPGSTAPPAHGVTSAPDTRPA with cysteine at the N-terminal end, and used the cysteine for chemical conjugation to keyhole limpet haemocyanin (KLH). Six breast cancer patients immunized with this conjugate plus the immunological adjuvant QS-21 have all produced high titre (by ELISA) IgG and IgM antibodies against the 30 aa MUC1 peptide, but these sera reacted moderately, or not at all, with MUC1-positive tumour cells. To understand this specificity better, we prepared a series of smaller peptides to determine the epitopes recognized by these immune sera in inhibition assays. Only peptides containing APDTRPA at the C-terminal end were able to completely inhibit ELISA reactivity for the full 30 aa peptide. No sera were completely inhibited by APDTR, APDTRP, PDTRPA or any other peptides that did not contain the full APDTRPA epitope. Remarkably, sera from all six patients recognized this same epitope and were completely inhibited by only this epitope. The specificity of these sera (1) primarily for C-terminal APDTRPA, and the absence of this epitope at the C-terminal end of any tumour mucins, and (2) the N-terminal APDTRPA alanine, which is normally buried in the beta turn MUC1 assumes in its secondary structure may explain the moderate to weak reactivity of these high titer sera against MUC1-positive tumour cells.