In the last five years, green fluorescent protein (GFP) has emerged from being a mere curiosity to become a reliable tool for molecular pharmacological research. GFP produces an intense and stable green fluorescence noncatalytically by absorbing blue light maximally at 395 nm and emitting green light with a peak at 509 nm. It consists of 238 amino acids and its molecular mass is 27-30 kDa. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence to be utilised in nonnative organisms, wherein it can be used as a reporter. This use of GFP permits real-time analysis of receptor dynamics. The emitted fluorescence can be used as a nontoxic marker and detected using fluorescence-activated cell sorting (FACS), thus avoiding any staining procedure, expensive mRNA analysis or hazardous radiolabeled binding assays. The potential value of GFP has also been recognized in orphan receptor research, where various GFP-tagged therapeutic proteins have been constructed in an attempt to identify the endogenous ligand(s). These chimeric proteins have been used to determine the site and time course of receptor expression and to relate receptor dynamics with therapeutic outcome. The preparation of new GFP constructs for identifying germ layer cells (endodermal, ectodermal, and mesodermal), as well as neuronal, haematopoietic, endothelial, and cartilage cells, has provided a useful battery of tissue/receptor-specific screening assays for new chemical entities. Genetically engineered cells with GFP expression have provided a valuable tool for automated analysis, and can be adapted for high-throughput systems. GFP is being increasingly utilised for the study of receptor dynamics, where, having already proved beneficial, it will likely continue to contribute towards the search for new classes of drugs, as well as to "de-orphaning" orphan receptors.

Animals↗

PubMedDate not supplied

Role of sympathetic nervous system in experimental hypertension and diabetes mellitus.

An investigation has been made to elucidate the role of sympathetic nervous system (SNS) in the development of renal hypertension and hyperglycemia in rats and rhesus monkeys using 6-hydroxydopamine (6-OHDA). Development of renal hypertension was blocked in weanling rats (80%), demedullated adult rats with 6-OHDA (75%) but not in adult rats sympathectomised with 6-OHDA. Weanling rats treated with 6-OHDA did not have any detectable catecholamine stores when measured 60 days after treatment with 6-OHDA. Unlike the weanling rats the hearts of the adult rats showed significant refilling of catecholamines 60 days after 6-OHDA treatment. 6-OHDA treatment and adrenalectomy did not modify the development of streptozotocin (STZ) induced hyperglycemia and the hypoglycemic effect of tolbutamide and phenformin. Chemical sympathectomy with 6-OHDA did not show any glucose intolerance in rhesus monkeys or any effect or insulin release. Phentolamine (PHE) and oxprenolol (OXP) pretreatment had no effect on development and maintenance of hyperglycemia. Although there was no effect on the endogenous catecholamines stored in the heart and brain in the diabetic rats, there was a marked increase in the urinary excretion of norepinephrine (NE), epinephrine (E), 3-methoxy-4 hydroxy mandelic acid (VMA) and creatinine. The rate of disappearance of (3H)-NE from rat heart was higher when compared to age matched controls. Diabetic rats showed increase responsiveness to angiotensin II and developed hypertension faster when their renal arteries were clamped as compared to controls. It is concluded that the functional SNS is important in the development of renal hypertension in the rat. Further the SNS in the rats and rhesus monkeys does not play any significant role in the STZ induced hyperglycemia, glucose intolerance and insulin release.

Adrenalectomy↗

PubMedDate not supplied

Sintamil -a new dibenzoxazepine antidepressant.

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Animals↗