Prabhavathi Fernandes

Handbook of Drug Screening

Key Factors for Successful High-Throughput Screening, John G. Houston Critical Components in High-Throughput Screening: Challenges and New Trends, Litao Zhang, Martyn N. Banks, and John G. Houston Hit-to-Probe-to-Lead Optimization Strategies: A Biology Perspective to Conquer the Valley of Death, Anuradha Roy, Byron Taylor, Peter R. McDonald, Ashleigh Price, and Rathnam Chaguturu Signal Detection Platforms for Screening in Drug Discovery, Ramakrishna Seethala Proteomic Analysis in Drug Discovery, Haiteng Deng, Yang Xu, and Linqi Zhang Screening and Characterization of G-Protein-Coupled Receptor Ligands for Drug Discovery, Ge Zhang and Mary Ellen Cvijic Nuclear Hormone Receptor Screening in Drug Discovery, Ramakrishna Seethala and Litao Zhang Emerging Novel High-Throughput Screening Technologies for Cell-Based Assays, Ilona Kariv, Alexander A. Szewczak, Nathan W. Bays, Nadya Smotrov, and Christopher M. Moxham In Vitro Strategies for Ion Channel Screening in Drug Discovery, Ravikumar Peri, Mark Bowlby, and John Dunlop Wheat from Chaff: General andMechanistic Triage of Screening Hits for Enzyme Targets, Mark R. Harpel Protein Kinases and Phosphatases, Pirthipal Singh MicroRNA Strategies in Drug Discovery, Wishva B. Herath, Dwi S. Karolina, Arunmozhiarasi Armugam, and Kandiah Jeyaseelan Strategies for Screening of Biologic Therapeutics, Ian Foltz and Francesca Civoli Cryopreserved Cells in Functional Cell-Based HTS Assays, Geetha Shankar and Kirk McMillan High-Content Screening with a Special Emphasis on Cytotoxicity and Cell Health Measurementsn Ralph J. Garippa and Ann F. Hoffman Effective Application of DrugMetabolism and Pharmacokinetics in Drug Discovery, Sharon Diamond and Swamy Yeleswaram Compound Management for Drug Discovery: An Overview, Moneesh Chatterjee and Martyn N. Banks Practical Approach to Quantitative High Throughput Screening, Wei Zheng, Ke Liu, and James Inglese Enabling the Large-Scale Analysis of Quantitative High-Throughput Screening Data, Noel T. Southall, Ajit Jadhav, Ruili Huang, Trung Nguyen, and Yuhong Wang Application of Nanobiotechnologies for Drug Discovery, K. K. Jain Index

Molecular Genetic Analysis of a Human Neuropeptide Y Receptor THE HUMAN HOMOLOG OF THE MURINE “Y5” RECEPTOR MAY BE A PSEUDOGENE

Neuropeptide Y is a 36-amino-acid peptide amide with numerous biological activities. These functions are mediated through several pharmacologically distinct receptors. To date five receptor subtypes have been cloned. Here we report the isolation, by low stringency homology cloning from a hypothalamic library, of a cDNA encoding the human homolog of the murine neuropeptide Y receptor subsequently reported (1). Translation of the human Y1-like receptor clone suggested that it encoded a receptor which is truncated in the third extracellular loop. Comparison of the human Y1-like sequence to that of the human Y1 receptor suggested that the truncated receptor could have resulted from a frameshift due to a single nucleotide deletion in the sixth transmembrane domain. Southern blot analysis suggested that the gene is single copy in the human genome. The gene is located on chromosome 5q. To test the hypothesis that allelic variation of nucleic acid length within the sixth transmembrane domain of the Y1-like receptor may exist to produce a functional receptor, genomic DNA from 192 individuals of various ages, ethnic backgrounds, and degrees of obesity were analyzed electrophoretically and by direct sequencing. No variation was detected in any of the subjects, indicating that this receptor subtype may be a transcribed pseudogene in humans.

Melatonin Agonists Induce Phosphoinositide Hydrolysis in Xenopus Laevis Melanophores

Melatonin, the principal hormone of the vertebrate pineal gland, has been implicated in a variety of neurobiological processes such as circadian rhythmicity and reproductive function. One of the earliest described actions of melatonin was its ability to cause pigment translocation in the dermal melanophores of amphibians. Melatonin binding sites have been identified in the brain of many species and in pigmented tumour cell lines; however, the dermal melanophores of the frog Xenopus Laevis possess the highest known density of melatonin binding sites. These cells are the source from which a melatonin receptor has been cloned and provide an excellent model to study melatonin-mediated signal transduction in an isolated cell system. In Xenopus melanophores, melatonin induces a rapid perinuclear aggregation of intracellular pigment which is associated with a pertussis toxin-sensitive inhibition of cAMP. We have previously demonstrated that a subtype of melatonin binding sites found in selected regions of the pigeon brain and in Syrian Hamster RPMI 1846 melatonin cells are functionally coupled to phosphoinositide hydrolysis as a second messenger. Here we now present evidence to suggest that Xenopus Laevis melanophores also possess melatonin binding sites which are functionally linked to phosphoinositide hydrolysis. Melatonin agonists induced phosphoinositide hydrolysis in melanophores in a concentration-dependent manner with a rank order of potency of 2-iodomelatonin > 6-chloromelatonin > N-acetylserotonin > melatonin. Stimulatory response of 2-iodomelatonin was blocked by the melatonin antagonist N-acetyltryptamine and the alpha-adrenergic antagonist prazosin, which has been shown to have high affinity for melatonin binding sites. Phosphoinositide hydrolysis induced by melatonin agonists was not blocked by the serotonin antagonist ketanserin or by phentolamine, an alpha-adrenergic antagonist, indicating that the response observed was not due to stimulation of 5-HT2a/2c receptors or alpha-adrenergic receptors. Furthermore, incubation of melanophores with the non-hydrolyzable G-protein source GTP-gamma-S attenuated the phosphoinositide dose response induced by 2-iodomelatonin, and pre-incubation of the cells with pertussis toxin had no effect on 2-iodomelatonin-induced phosphoinositide hydrolysis. The present data suggest that Xenopus Laevis Melanophores possess G-protein linked pertussis toxin-insensitive melatonin binding sites which are functionally coupled to phosphoinositide hydrolysis as a signal transduction mechanism.

Interaction of NT-4 and BDNF with gp145trkb receptor: effect on cellular metabolism.

In the present study a silicon microphysiometer (Cytosensor) was applied in investigating interactions of gp145(trkb), a member of the tyrosine kinase receptor family, with different neurotrophic factors. NIH-3T3 cells transfected with gp145(trkb) receptors (NIH3T3/trkB cells) were utilized in the studies. Treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3) induced changes in the metabolic rate of NIH3T3/trkB cells. In contrast, no response was observed with nerve growth factor (NGF). The effects of NT-4 and BDNF on NIH3T3/trkB cells were receptor-specific in that they did not induce metabolic rate changes in wild type NIH3T3 cells or cells transfected with either gp140(trkb) (TrkA) or gp145(trkb) (TrkC) receptors. In contrast, NT-3 induced metabolic rate changes in cells transfected with each of the three different Trk receptors. The activity of NT-4 was significantly higher than that of BDNF. K252a, a protein kinase inhibitor, reduced the NT-4- and BDNF-induced response of the NIH3T3/trkB cells. This suggests that the NT-4 and BDNF-induced metabolic rate changes are associated with autophosphorylation of the tyrosine protein kinase residues. This hypothesis is further supported by results of western blot analysis. The results show that interactions of Trk receptors with neurotrophic factors result in metabolic changes in cells expressing the receptors.