Detection of Protein Bound Volatile Compounds in Buffalo Urine

Animal urine contains different non-polar volatile compounds, which are known to stimulate their sexual behavior. These compounds collectively termed as pheromones that remain bound to some urinary proteins, which help in their signaling. The objective of this experiment was to identify the urinary volatile compounds before and after protease treatment in bull and in various reproductive stages of female buffaloes, viz. estrus, diestrus and pregnancy, by chemical extraction followed by thin layer chromatography (TLC). Buffalo bull urine showed different compounds in TLC upon protease treatment, where as no change in retention time values were observed for female buffalo urine upon protease treatment. It was found that buffalo bull urine contains protein bound non-polar compounds, which can be set free upon protease treatment and detected by TLC

Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P <0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

316-320Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P <0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Immunophenotyping of non-Hodgkin’s lymphoma by flowcytometry on fine needle aspiration

Background: Lymphoma represents one of the major health problems all over the world. Flow cytometry (FCM) can be used on fine-needle aspiration cytology (FNAC) from lymph node as an ancillary technique. Aim of the study was to assess the utility of flowcytometry (FCM) in diagnosis and differentiation of reactive hyperplasia and non-Hodgkin’s lymphoma (NHL) on FNAC.Methods: The study was carried out on 50 cases, 25 each of reactive hyperplasia and suspicious or confirmed NHL on FNAC. FCI was performed with a complete panel of antibodies on FACS Canto II FCM.Results: All 25 cases of reactive hyperplasia on FNAC were polyclonal on FCM. FCM could be performed in 22 cases (88%) out of 25 suspicious NHL and in three cases the material was inadequate on aspirate. Out of 22 cases of NHL on FNAC 17 cases (77.30%) were diagnosed as B-NHL on FCM. Light chain restriction was demonstrated in 15/17 cases. With the help of FCI, 6 cases were diagnosed as small cell lymphocytic lymphoma, one case as mantle cell lymphoma, one case as follicular lymphoma, and 9 cases as B-NHL-NOS. Histopathology diagnosis was available in nine cases and were in concordance to FCM. Sensitivity of combined FNAC and FCM in sub-classification was 77.30% (17/22). Four cases showed discordance between FNAC and FCM.Conclusion: We concluded that FCM enhances the diagnostic ability of FNAC, playing a crucial role in a rapid and accurate differential diagnosis between reactive hyperplasia, B-NHL and T-NHL

EFFECT OF HEAT STRESS IN TROPICAL LIVESTOCK AND DIFFERENT STRATEGIES FOR ITS AMELIORATION

Stress is a broad term, generally used in negative connotation and is described as the cumulative detrimental effect of a variety of factors on the health and performance of animals. Heat stress occurs in animals when there is an imbalance between heat production within the body and its dissipation. Heat stress is one of the wide varieties of factors which causes oxidative stress in-vivo. Reactive oxygen species (ROS), the major culprits for causing oxidative stress, are constantly generated in vivo as an integral part of metabolism. ROS may cause oxidative stress when their level exceeds the threshold value. They trigger progressive destruction of polyunsaturated fatty acids (PUFA), ultimately leading to membrane destruction. Body employs antioxidants to quench these free radicals. The enzymatic antioxidants like superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) act by scavenging both intracellular and extracellular superoxide radical and preventing lipid peroxidation of plasma membrane. Non-enzymatic antioxidants include vitamins like vitamins C, A and E, proteins like albumin, transferrin, glutathione (GSH) etc. Antioxidant nutrient supplementation especially vitamins C, A and E, zinc and chromium can be used to attenuate the negative effects of environmental stress

Methanolic extract of Phlogacanthus thyrsiflorus Nees leaf induces apoptosis in cancer cells

153-161Phlogacanthus thyrsiflorus Nees is a medicinal herb commonly used in traditional folk medicine, and it is known to possess anticancer activity. Here, we explored the anticancer properties of methanolic extract of P. thyrsiflorus leaves in HeLa and MCF-7 cell lines. We observed nuclear fragmentation as indication of apoptosis in the MPT treated cancer cells using haematoxylin and eosin (H&amp;E) as well as fluorescent dye. DNA from the treated cells showed characteristic laddering of DNA fragments in agarose gel electrophoresis. Cell populations undergoing secondary necrosis following apoptosis could also be detected in FACS by annexin V/propidium iodide (PI) staining. Activated caspase-3 in the treated HeLa cells was detected by polyclonal anti-caspase-3 antibody utilizing immunocytochemistry. Using transmission electron microscopy, sub-cellular changes like rough endoplasmic reticulum, swollen mitochondria, distorted mitochondrial membrane, loss of cristae and matrix were observed in the treated HeLa cells. Extensive plasma membrane blebbing was also observed by scanning electron microscopy. Our findings support that Phlogacanthus thyrsiflorus leaves are natural source of potent anticancer agent

Methanolic extract of Phlogacanthus thyrsiflorus Nees leaf induces apoptosis in cancer cells

Phlogacanthus thyrsiflorus Nees is a medicinal herb commonly used in traditional folk medicine, and it is known to possess anticancer activity. Here, we explored the anticancer properties of methanolic extract of P. thyrsiflorus leaves in HeLa and MCF-7 cell lines. We observed nuclear fragmentation as indication of apoptosis in the MPT treated cancer cells using haematoxylin and eosin (H&amp;E) as well as fluorescent dye. DNA from the treated cells showed characteristic laddering of DNA fragments in agarose gel electrophoresis. Cell populations undergoing secondary necrosis following apoptosis could also be detected in FACS by annexin V/propidium iodide (PI) staining. Activated caspase-3 in the treated HeLa cells was detected by polyclonal anti-caspase-3 antibody utilizing immunocytochemistry. Using transmission electron microscopy, sub-cellular changes like rough endoplasmic reticulum, swollen mitochondria, distorted mitochondrial membrane, loss of cristae and matrix were observed in the treated HeLa cells. Extensive plasma membrane blebbing was also observed by scanning electron microscopy. Our findings support that Phlogacanthus thyrsiflorus leaves are natural source of potent anticancer agent

Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Abstract Background Cholesterol in lipid raft plays crucial role on cancer cell survival during metastasis of cancer cells. Cancer cells are reported to enrich cholesterol in lipid raft which make them more susceptible to cell death after cholesterol depletion than normal cells. Methyl-β-cyclodextrin (MβCD), an amphipathic polysaccharide known to deplete the membrane cholesterol, induces cell death selectively in cancer cells. Present work was designed to identify the major form of programmed cell death in membrane cholesterol depleted cancer cells (MDA-MB 231 and 4T1) and its impact on migration efficiency of cancer cells. Methods Membrane cholesterol alteration and morphological changes in 4T1 and MDA-MB 231 cancer cells by MβCD were measured by fluorescent microscopy. Cell death and cell proliferation were observed by PI, AO/EB and MTT assay respectively. Programme cell death was confirmed by flow cytometer. Caspase activation was assessed by MTT and PI after treatments with Z-VAD [OME]-FMK, mitomycin c and cycloheximide. Necroptosis, autophagy, pyroptosis and paraptosis were examined by cell proliferation assay and flow cytometry. Relative quantitation of mRNA of caspase-8, necroptosis and autophagy genes were performed. Migration efficiency of cancer cells were determined by wound healing assay. Results We found caspase independent cell death in cholesterol depleted MDA-MB 231 cells which was reduced by (3-MA) an autophagy inhibitor. Membrane cholesterol depletion neither induces necroptosis, paraptosis nor pyroptosis in MDA-MB 231 cells. Subsequent activation of caspase-8 after co-incubation of mitomycin c and cycloheximide separately, restored the cell viability in cholesterol depleted MDA-MB 231 cells. Down regulation of caspase-8 mRNA in cholesterol depleted cancer cells ensures that caspase-8 indirectly promotes the induction of autophagy. In another experiment we have demonstrated that membrane cholesterol depletion reduces the migration efficiency in cancer cells. Conclusion Together our experimental data suggests that membrane cholesterol is the crucial for the recruitment and activation of caspase-8 as well as its non-apoptotic functions in cancer cells. Enriched cholesterol in lipid raft of cancer cells may be regulating the cross talk between caspase-8 and autophagy machineries to promote their survival and migration. Therefore it can be explored to understand and address the issues of chemotherapeutic and drugs resistance

Subacute toxicity of anilofos, a new organophosphorus herbicide in male rats: Effect on lipid peroxidation and ATPase activity

1113-1117Effects of anilofos on lipid peroxidation — an index of oxidative stress, ATPase activity —  an integral part of active transport mechanisms for cations, GSH level and GST activity were evaluated in blood (erythrocyte/plasma), brain and liver of male rats after daily oral exposure to 50, 100 or 200 mg/kg for 28 days. None of the doses increased lipid peroxidation. The lowest dose, rather, produced marginally significant decrease in peroxidation in liver. Different doses of anilofos decreased GSH content and activities of GST and ATPases. Inhibition of total ATPase (34-44%) and Na+-K+-ATPase (45-52%) activities was maximum in liver, while that of Mg2+-ATPase (46-56%) was more in erythrocyte. Results indicate that anilofos may not cause oxidative damage to cell membrane in repeatedly exposed animals and may cause neuronal/cellular dysfunction by affecting ionic transport across cell membrane.</span

MOESM2 of Depletion of membrane cholesterol compromised caspase-8 imparts in autophagy induction and inhibition of cell migration in cancer cells

Additional file 2: Figure S2. Cholesterol depletion induced cell death in various cell lines. [a] cholesterol depletion induced cell death. MDA-MB 231, 4T1 and Balbc3T3 Cell lines were treated with different concentration of MβCD for 24 h. Cell viability was measured by MTT assay. 2[b] Vero, MDCK, 4T1, Balb/c3T3 and MDA-MB 231 cells were exposed to the 5mM MβCD for 16 h and cell death were measured by MTT assay. Statistical analysis: One way anova, post hock test Tukey. P*<0.05 P**<0.01, P**<0.001, N.S.-Not significant