Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immunerelated genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus–cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture

“Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation

Unveiling the molecular and regulatory mechanisms that prevent in vitro transformation in shrimp remains elusive in the development of continuous cell lines, with an arduous history of over 25 years (Jayesh et al., 2012). Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. In addition, the regulatory mechanisms that prevent in vitro transformation and carcinoma in shrimps might provide new leads for the development of anti-ageing and anti-cancer interventions in human (Vogt, 2011) and in higher vertebrates. This highlights the importance of developing shrimp cell lines, to bring out effective prophylactics against shrimp viruses and for understanding the mechanism that induce cancer and ageing in human.. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. With this backdrop, the research described in this thesis attempted to develop molecular tools for induced in vitro transformation in lymphoid cells from Penaeus monodon and for the development of continuous cell lines using conventional and novel technologies to address the problems at cellular and molecular level.

Development of a cell culture system from the ovarian tissue of African catfish Clarias gariepinus

A growth medium with Leibovitz-15 L-15.as the base, supplemented with foetal bovine serum 10% vrv., fish muscle extract 10% vrv., prawn muscle extract 10% vrv., lectin concanavalin A. 0.02 mg mly1., lipopolysaccharide 0.02 mg mly1., glucose D 0.2 mg mly1., ovary extract 0.5% vrv.and prawn haemolymph 0.5%. has been formulated with 354"10 mOsm for the development and maintenance of a cell culture system from the ovarian tissue of African catfish, Clarias gariepinus. For its subculturing, a cell dissociationrextracting solution, composed of equal portions of trypsin phosphate versene glucose TPVG. containing 0.0125% wrv.trypsin and 25% vrv.non-enzymatic cell dissociation solution 1 and 2, has also been developed with which the cell culture can be passaged 15 times after which they cease to multiply and consequently perish. The cell cultures can be maintained for 12–15 days without fluid change between the passages. This is the first report of a cell culture system from the ovarian tissues of African catfish

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 % salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg-1 and temperature of incubation was 25 8C. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (29), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation ‘‘cocktail’’.

DOPAMINE D2 RECEPTOR FUNCTIONAL REGULATION : cAMP AND IP3 ROLE IN PANCREATIC REGENERATION

The present study deals with the differential regulation of Dopamine content in pancreas and functional regulation of Dopamine D2 receptor in brain regions such as hypothalamus, brain stem, cerebral cortex and corpus striatum play an important role during pancreatic islets cell proliferation and insulin secretion. Though may reports are there implicating the functional interaction between DA receptor and pancreatic islets cell insulin secretion, the involvement of specific DA D2 receptors and changes in second messenger system during insulin secretion and pancreatic islets cell proliferation were not given emphasis. Down regulation of DA content in brain regions and pancreatic islets were observed during pancreatic regeneration. Up regulation of DA content in plasma and adrenals down regulated sympathetic activity in pancreas which cause an increase in insulin secretion and pancreatic islets cell proliferation during pancreatic regeneration. There was a differential regulation of DA D2 receptor in brain regions. The pancreatic islets DA D2 receptors were lip regulated during pancreatic regeneration. DA D2 receptor activation at specific concentration has accounted for increased pancreatic islets cell proliferation. In vitro experiments have proved the differential regulation of DA on insulin synthesis and pancreatic islets cell proliferation. Inhibitory effect of DA on cAMP and stimulatory effect of DA on IP3 through DA D2 receptors were observed in in vitro cell culture system. These effects are correlating with the DA, cAMP and IP3 content during pancreatic regeneration and islets cell proliferation. Up regulation of intracellular Ca2+ was also observed at 10-8 M DA, a specific concentration of DA which showed maximum increase of IP3 content in pancreatic islets through DA D2 receptor activation in in vitro culture. These in vitro data was highly correlating with the changes in DA, cAMP and IP3 content in pancreas during pancreatic regeneration and insulin secretion. Thus we conclude that there is a differential functional regulation of DA and DA D2 receptors in brain and pancreas during pancreatic regeneration. In vitro studies confirmed a concentration depend functional regulation of DA through DA D2 receptors on pancreatic islets cell proliferation and insulin secretion mediated through increased cAMP, IP3 and intracellular Ca2+ level. This will have immense clinical significance in the management in diabetes mellitus.

Studies On In Vitfio Production Of Secondary Metabolites From Medicinal Plants

In the light of the very huge demand for natural ephedrine and pseudoephidrine, a search for an angiosperm plant containing the alkaloid ephedrine was made and could locate Sida spp. of malvaceae family. Sida is a large genus of, herbs and shrubs distributed throughout the tropics. About a dozen species occur in India. The medicinally important species known are S.rhombrfolia S.cordata and S.spinosa (Anon, 1972). Among the various species, S.rh0mbIfolia is the most widely used one in the traditional system of medicine. An attempt was made in the present study to develop an ideal bioprocess for the in vitro production of ephedrine from the cell culture system of Sida rhombrfolia Linn. ssp. retusa. The callus and suspension culture were initiated and attempts were made to enhance the yield positively by employing various strategies like mutagenesis, immobilization and addition of precursors, elicitors and penneabilizing agents.

Gaba Receptor Gene Expression during Rat Liver Cell Proliferation and Its Function in Hepatocyte Cultures

The present thesis is an attempt to understand the role of GABA, GABAA and GABAB receptors in the regulation of liver cell proliferation using in vivo and in vitro models. The work also focuses on the brain GABAergic changes associated with normal and neoplastic cell growth in liver and to delineate its regulatory function. The investigation of mechanisms involving mitogenic models without cell necrosis may contribute our knowledge about both on cell growth, carcinogenesis, liver pathology and treatment. Objectives of the present study are, to induce controlled liver cell proliferation by partial hepatectomy and lead nitrate administration and uncontrolled cell proliferation by N-nitrosodiethylamine treatment in male Wistar rats, the changes in the content of GABA, GABAA,GABAB in various rat brain regions. To study the GABAA and GABAB receptor changes in brain stem, hypothalamus, cerebellum and cerebral cortex during the active cortex during the period of active DNA synthesis in liver of different experimental groups. The changes in GABAA and GABAB receptor function of the brain stem, hypothalamus and cerebellum play an important role sympathetic regulation of cell proliferation and neoplastic growth in liver. The decrease in GABA content in brain stem, hypothalamus and cerebellum during regeneration and neoplasia in liver. The time course of brain GABAergic changes was closely correlated with that of heptic DNA synthesis. The functional significance of these changes was further explored by studying the changes in GABAA and GABAB receptors in brain.