Gautam Kaul

Eukaryotic elongation factor-2 (eEF2): its regulation and peptide chain elongation.

Regulation at the level of translation in eukaryotes is feasible because of the longer lifetime of eukaryotic mRNAs in the cell. The elongation stage of mRNA translation requires a substantial amount of energy and also eukaryotic elongation factors (eEFs). The important component of eEFs, i.e. eEF2 promotes the GTP‐dependent translocation of the nascent protein chain from the A‐site to the P‐site of the ribosome. Mostly the eEF2 is regulated by phosphorylation and dephosphorylation by a specific kinase known as eEF2 kinase, which itself is up‐regulated by various mechanisms in the eukaryotic cell. The activity of this kinase is dependent on calcium ions and calmodulin. Recently it has been shown that the activity of eEF2 kinase is regulated by MAP kinase signalling and mTOR signalling pathway. There are also various stimuli that control the peptide chain elongation in eukaryotic cell; some stimuli inhibit and some activate eEF2. These reports provide the mechanisms by which cells likely serve to slow down protein synthesis and conserve energy under nutrient deprived conditions via regulation of eEF2. The regulation via eEF2 has also been seen in mammary tissue of lactating cows, suggesting that eEF2 may be a limiting factor in milk protein synthesis. Regulation at this level provides the molecular understanding about the control of protein translocation reactions in eukaryotes, which is critical for numerous biological phenomenons. Further the elongation factors could be potential targets for regulation of protein synthesis like milk protein synthesis and hence probably its foreseeable application to synthetic biology. Copyright

Calcium requirement and time course of capacitation of goat spermatozoa assessed by chlortetracycline assay

Summary We standardized chlortetracycline fluorescent assay for studies of calcium requirement and time course of capacitation of goat spermatozoa. Three distinct fluorescent patterns were easily detected in goat spermatozoa incubated under capacitating conditions. Categorised according to nomenclature reported earlier, these are: ‘F’ with bright fluorescence in the post‐acrosomal region, characteristic of uncapacitated acrosomal‐intact cells; ‘B’ with bright fluorescence on the anterior portion of the head and dark band in the postacrosomal region, characteristic of capacitated, acrosome‐intact cells; ‘AR’ with lack of fluorescence on the head characteristic of acro‐some‐reacted cells. A close correspondence was observed when the results of CTC assay were compared with those obtained by transmission electron microscopy.

Role of Heat Shock Proteins in Diseases and Their Therapeutic Potential

Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaperones in processes like protein folding and protein trafficking between different intracellular compartments. They are induced during stress conditions like oxidative stress, nutritional deficiencies and radiation. They are released into extracellular compartment during necrosis. However, recent research findings highlights that, they are not solely present in cytoplasm, but also released into extracellular compartment during normal conditions and even in the absence of necrosis. When present in extracellular compartment, they have been shown to perform various functions like antigen presentation, intercellular signaling and induction of pro-inflammatory cytokines. Heat shock proteins represents as dominant microbial antigens during infection. The phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins has led to proposition that, microbial heat shock proteins can induce self reactivity to host heat shock proteins and result in autoimmune diseases. The self-reactivity of heat shock proteins protects host against disease by controlling induction and release of pro-inflammatory cytokines. However, antibodies to self heat shock proteins haven been implicated in pathogenesis of autoimmune diseases like arthritis and atherosclerosis. Some heat shock proteins are potent inducers of innate and adaptive immunity. They activate dendritic cells and natural killer cells through toll-like receptors, CD14 and CD91. They play an important role in MHC-antigen processing and presentation. These immune effector functions of heat shock proteins are being exploited them as therapeutic agents as well as therapeutic targets for various infectious diseases and cancers.

Health hazards associated with nanomaterials

Nanotechnology is a major scientific and economic growth area and presents a variety of hazards for human health and environment. It is widely believed that engineered nanomaterials will be increasingly used in biomedical applications (as therapeutics and as diagnostic tools). However, before these novel materials can be safely applied in a clinical setting, their toxicity needs to be carefully assessed. Nanoscale materials often behave different from the materials with a larger structure, even when the basic material is same. Many mammals get exposed to these nanomaterials, which can reach almost every cell of the mammalian body, causing the cells to respond against nanoparticles (NPs) resulting in cytotoxicity and/or genotoxicity. The important key to understand the toxicity of nanomaterials is that their minute size, smaller than cellular organelles, allows them to penetrate the basic biological structures, disrupting their normal function. There is a wealth of evidence for the noxious and harmful effects of engineered NPs as well as other nanomaterials. The rapid commercialization of nanotechnology field requires thoughtful, attentive environmental, animal and human health safety research and should be an open discussion for broader societal impacts and urgent toxicological oversight action. While ‘nanotoxicity’ is a relatively new concept to science, this comprehensive review focuses on the nanomaterials exposure through the skin, respiratory tract, and gastrointestinal tract and their mechanism of toxicity and effect on various organs of the body.

Ultrasound Guided Transplantation of Enriched and Cryopreserved Spermatogonial Cell Suspension in Goats

Spermatogonial stem cells transplantation provides a unique approach for studying spermatogenesis. Initially developed in mice, this technique has now been extended in farm animals and provides an alternative means to preserve valuable male germ line and to produce transgenic animals. The aim of this study was to enrich type A spermatogonial cells amongst the isolated cells from goat testis, to cryopreserve these enriched populations of cells and their subsequent transplantation in unrelated recipient goats under ultrasound guidance. The cells were isolated enzymatically and enriched by differential plating and separation on discontinuous percoll gradient. Ultrasound guided injection of trypan blue dye into rete testis resulted in 20-30% filling of the seminiferous tubules. Prior to transplantation, the cells were labelled with a fluorescent dye to trace donor cells in recipient seminiferous tubules after transplantation. The fluorescent-labelled cells were observed up to 12 weeks after transplantation.

Toxicity of titanium oxide nanoparticles causes functionality and DNA damage in buffalo (Bubalus bubalis) sperm in vitro.

The present study has examined the effect of different concentrations (1 μg/ml, 10 μg/ml and 100 μg/ml) of titanium oxide (TiO2) nanoparticles (NPs) (<100 nm) on viability, membrane integrity, capacitation status and DNA integrity of buffalo spermatozoa. Characterization of NPs was done by the transmission electron microscopy (TEM) and dynamic light scattering (DLS). Sperm chromatin dispersion (SCD) test and acridine orange test (AOT) were employed to detect DNA fragmentation in sperm treated with NPs. There was significant (p < 0.05) decrease in cell viability and membrane integrity (assessed by enzyme leakage) at 6 h of incubation with NPs. However, significant (p < 0.05) increase in sperm capacitation was observed for TiO2 NP albeit at lower concentrations. In DNA fragmentation assay, there was dose-dependent increase in the DNA fragmentation (r = 0.96). Ultrathin cross-sections revealed TiO2 NPs inside head and plasma membrane of the buffalo spermatozoa as assessed by TEM. These studies suggest that TiO2 NPs may have cytotoxic effect on buffalo spermatozoa by affecting sperm functionality and causing high amount of DNA fragmentations.

Isolation and enrichment of type A spermatogonia from pre-pubertal buffalo ( Bubalus bubalis) testis

The aim of this study was to isolate and subsequently enrich type A spermatogonia from pre‐pubertal buffalo testis. Two‐step enzymatic digestion was used to isolate spermatogonia from 10 to 14 months pre‐pubertal buffalo calves, resulting in maximal release of spermatogonia from the seminiferous tubules. After enzymatic digestion, the type A spermatogonia were subsequently enriched by differential plating and Percoll gradient centrifugation. The identity of type A spermatogonia was determined by light microscopy and further characterised by Dolichos biflorus agglutinin, a specific marker for bovine type A spermatogonia by fluorescence‐activated cell sorting analysis. After enzymatic isolation, the cell suspension contained about 27% of type A spermatogonia, which was enriched up to 71% with >70% cell viability. Further flow cytometric analysis showed the presence of THY1+ cells (cells expressing thymocyte differentiation antigen 1), suggesting that THY1 is a conserved marker of the undifferentiated spermatogonial cells in buffalo. The isolation of the enriched type A spermatogonia from buffalo testis opens ways to study the further biochemical characteristics of this important class of germ cells in this species.

Assessment of buffalo (Bubalus bubalis) sperm DNA fragmentation using a sperm chromatin dispersion test.

Chromosomal fragmentations or damage in sperm DNA has considerable value in determination of semen quality. However, rapid and/or simple method to assess sperm DNA integrity in buffalo has apparently not been reported. In the present study, SCD was used for the first time in buffalo bulls for assessment of sperm DNA fragmentation. A modified SCD protocol, under bright field microscope was developed and validated by comparison with other routine tests which can be used for processing of samples. The DNA fragmentation index (DFI) from SCD was correlated with semen quality parameters viz. viability (r=-0.68, p<0.05), membrane integrity (r=-0.74, p<0.05) and capacitation status (r=-0.69, p<0.05). The amount of DNA fragmentation assessed by SCD was highly correlated (R=0.874, p<0.05) with results of acridine orange test (AOT), a traditional method of assessing DNA damage. There were no significant differences between two observers with regards to scoring dispersion patterns. Therefore, the SCD test can be routinely used for detection of DNA fragmentation in buffalo sperm, with potential for replacing conventional time consuming tests.

Cereulide and diarrheal toxin contamination in milk and milk products: a systematic review

Abstract Emetic and diarrheal toxins produced by endospore-forming Bacillus cereus causes a huge loss to dairy industry. Milk is the suitable medium for the growth of B. cereus. The pathogen is of particular concern in the dairy industry because in spite of aggressive cleaning practices performed by the dairy industry, it is impossible to destroy these hydrophobic spores which adhere to the pipelines of the dairy-processing plant and further these spores may also form biofilms in the milk. The reporting rate of illness caused by B. cereus is vague, usually due to the short duration of (<24 h) the diarrheal and emetic syndromes. The study of toxins production by B. cereus isolates is essential to better determine the methods of controlling these toxins in dairy industry.

Inhibition of goat sperm-zona binding by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida

The zona pellucida (ZP) in a majority of species consists of three antigenic families with wide inter species cross reactivity. The glocoproteins of heat solubilized goat zona pellucida (gZP) were isolated into three families by high performance liquid chromatography on a gel filtration column. Their molecular identities were 180, 95 and 75 kDa for gZP1, gZP2 and gZP3 respectively. However under reducing conditions, four zona pellucida fractions of 135, 95, 75 and 42 kDa were observed. The monoclonal antisera Mab-5H4 against porcine ZP4 (a 23 kDa glycoprotein) easily recognized the 180 and 42 kDa gZP fractions. It was also found that the 5H4 antisera strongly inhibited homologous goat sperm-zona binding. Finally, using immunofluorescent staining the goat occytes reacted positively with the 5H4 antisera. The fluorescence was uniform at 1 : 10 antisera dilution but tended to fade at higher dilution’s. Results suggest that the gZP has an antigen epitope correlative to the 5H4 antisera which is involved in sperm-zona interaction and could be a good candidate for the development of a contraceptive vaccine. # 2000 Elsevier Science B.V. All rights reserved.