Research



Research Highlights
 
Application Oriented Research
 

Wildlife Management 
Based on the DNA and reproductive biology studies, the CCMB, in collaboration with the Chief Conservator of Forests, Government of Andhra Pradesh and Curator, Nehru Zoological Park, Hyderabad, has initiated a socially relevant project to set up a satellite facility for the conservation of endangered species with a view to create germplasm (sperm and egg) and tissue banks, and carry out research in the areas of in vitro fertilization, artificial insemination, cloning and molecular breeding of endangered species, such as tigers, lions and leopards. Named as the Laboratory for the Conservation of Endangered Species (LaCONES), this facility is being funded by the Central Zoo Authority of India and the Dept. of Biotechnology, New Delhi. 

Assessment of inbreeding depression in the Indian leopard, Panthera pardus:
Semen parameters such as volume, sperm density, and the number of motile and morphologically normal spermatozoa varied between individuals. Further, it was demonstrated that cryopreserved spermatozoa were motile and capable of penetrating zona free hamster oocytes. Application of computer-aided sperm analysis (CASA) to test the quality of the spermatozoa in the wild animals is being done to identify leopards with the best semen profiles for captive breeding programmes. 


Molecular characterization of scented rice germplasm of India 
CCMB has undertaken studies on the aromatic fine quality rices popularly known as basmati rices on a special request from the CSIR Headquarters with an aim to develop efficient technologies which can provide reliable genetic descriptors/ID cards of the germplasm. Today, India exports 0.3 to 0.5 million tonnes of the basmati rice, which is about two thirds of basmati rice produced in our country, earning about 15 billion rupees annually. The unsurpassed quality of basmati produced in the Indian subcontinent is a proprietary item of this region and needs to be protected not only by producing better varieties but also by way of claiming Intellectual Property Rights (IPR). Using DNA fingerprinting techniques, it is observed that the scored genetic variation can be used to distinguish basmati and non-basmati rices; the genetic variation in the Indian basmati lines is less; the Indian basmati germplasm is quite distinct from the other exotic aromatic rice germplasm; and the DNA markers can be reliably used to ascertain the hybrid status and seed purity of the germplasm. 


Tumour biology 
Mutations that lead to increased frequency of precise excision of transposon Tn10 were classified into two categories, depending on whether they did or did not also increase precise excision frequency of the mini-Tn10 derivatives. Mutations in uvrD were shown to be incompatible with lon because of chronic SOS induction, and the hypothesis has been advanced that the latter results because of a need for the UvrD helicase in unwinding secondary structures on the lagging strand immediately behind the progressing replication fork. CCMB has applied for a US patent (A process for identifying mutagens and antimutagens) based on these studies.This is repeated on pg 63 - highlighted in green


Cataract
Calcium homeostasis plays an important role in the physiology of cataract. Intracellular transglutaminase, a calcium-dependent enzyme catalyses the formation of epsilon -(gamma -glutamyl)-lysine isopeptide cross-links between polypeptides. Isolating N epsilon -(gamma -glutamyl) lysine peptides from cataractous specimens called attention to the role of transglutaminase in lens. Beta-crystallins are the known substrates for transglutaminase. CCMB scientists have identified alpha-crystallin as another substrate for this enzyme in the lens. There must exist calcium regulation systems in the lens, though there has been very little success in identifying them. Scientists have earlier shown the calcium binding properties of the eye lens protein beta -crystallin. Putative calcium-binding sites have been identified in the crystallin fold and it is suggested that the beta-gamma-crystallin superfamily represents a novel class of crystallin-like calcium binding proteins. 


Molecular and epidemiological characterization of clinical and environmental isolates of Acanthamoeba keratitis - a pathogenic protozoan causing corneal ulceration:
The genus Acanthamoeba belongs to free-living soil and water amoebae that are widespread in the environment and have the potential to cause many serious infectious diseases. In humans it causes keratitis, a sight threatening disease. CCMB, in collaboration with LV Prasad Eye Institute, Hyderabad, has initiated studies to standardize experimental protocols for DNA isolation and DNA typing of the isolates and also for detailed morphological characterization using standard microbiological techniques. For DNA typing and molecular characterization, 6 sets of genus-specific and pathogenic strain-specific primers have been designed and synthesized to ascertain the 18S rDNA sequence variation. Currently, optimum PCR conditions, sensitivity and specificity of these primers are being evaluated using DNA of isolates from human corneal patients and other reference isolates. 

Effect of ras p21 on GPI-anchored molecules in the protozoan parasite, Leishmania donovani: 
At least 50% of the surface of the Leishmania parasite is covered by two GPI-anchored molecules, gp63 and LPG, both of which are suspected to be involved in virulence mechanisms. CCMB was examining whether ras p21 can be used to perturb GPI-anchored molecules in Leishmania, and the results suggest that (a) expression of activated ras p21 in Leishmania causes a reduction in expression of cell surface molecules, (b) this effect of ras p21 appears to be directed against expression of specific molecules since the overall growth of the parasite remains unaffected, and (c) ras p21 expression seems to specifically affect the surface expression of GPI-anchored molecules (gp63 and LPG) and not that of non-GPI anchored molecules. 


Screening of bioactive peptides against Leishmania donovani:
Treatment for visceral leishmaniasis is presently inadequate and has harmful side effects. But naturally occurring peptides might hold promise for alternative strategies towards novel drug design. At least five out of the ten antibacterial peptides tested by the CCMB have shown a significant lytic effect on Leishmania promastigotes, with LD50s in µM range. While the mechanism of action of these peptides on the Leishmania cell is not clear; they appear to cause rapid lysis of the parasite. 

In addition, CCMB is developing a screening protocol for bioactive molecules effective against Leishmania based on the luciferase reporter gene activity as a rapid, machine-read assay for the viability of the parasite. 


Induction of elevated levels of interferon gamma (IFN gamma) in mice, using the T-lymphocyte triggering factor (TLTF) delivered as a DNA vaccine:
T-Lymphocyte Triggering Factor (TLTF) is a protein identified in African Trypanosomes, which has been demonstrated to induce CD8+ T cells to secrete IFNgamma . IFNgamma has been associated with immune responses against various intracellular pathogens. The objectives of this project are: firstly, to attempt to raise the levels of IFNgamma in mammals using the TLTF cDNA delivered as naked DNA; and secondly, to examine whether the triggering of the host immune system by such means offers any protection to the animal against intracellular infections. The results indicate that injecting TLTF DNA alone does not alter IFNgamma levels in animals. However, animals that first received doses of the TLTF cDNA and were then given an infection of Leishmania donovani, showed raised IFN-gamma levels in serum; secondly, splenocytes, from such animals, when re-stimulated with the same parasites, showed an enhanced level of IFN-gamma expression. In addition, although the TLTF cDNA has been injected into muscle tissue, it is the splenocytes, which exhibit IFN-gamma production, suggesting that the effect of the TLTF cDNA is systemic.