High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify ‘hit’ molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer anti-inflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

Metabolism of arachidonic acid in sheep uterus: in vitro studies.

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

Fatty acid profile and the atherogenic risk in fish consuming and non fish consuming people.

Epidemiological studies have indicated a lower incidence of cardiovascular disease in populations with a high habitual intake of marine fish and fish oils, and this apparent protection may be due to the presence of n-3 fatty acids in the dietary fish. Two populations, a rish consuming one from coastal area and an inland one with non fish consuming from Nellore district or South India were selected for the present study. A total of one thousand healthy subjects were randomly chosen (500 in each population) from both sexes, aged from 20 to 70 years. The concentration of serum cholesterol, triacylglycerol and LDL-cholesterol were significantly lower in fish consumers, whereas HDL-cholesterol was higher in fish consumers than in non fish consumers. The difference between the percent quantities of the three n-3 fatty acids (eicosapentaenoic, docosapentaenoic and docosahexaenoic acids) was highly significant (p < 0.001) with a greater value in fish consumers.

In vitro effects of gossypol and lactic acid on rat uterus and ovary during implantation and antiimplantation.

ATPase activity of uterus and ovary was markedly elevated in presence of gossypol and decreased in presence of lactic acid indicating activation and inhibition of energy metabolism by gossypol and lactic acid respectively. The elevated levels of glycogen in uterus indicate inhibition of glycogenolysis as supported by phosphorylase activity. Whereas in ovary the glycogen depletion indicates activation of glycogenolysis supported by phosphorylase activity. The activity levels of aldolase and G-6-PDH decreased in the uterus in presence of gossypol and increased in presence of lactic acid. The same were elevated in ovary indicating the activation of hexose mono and diphosphate pathways. Lactic acid accumulated in presence of both gossypol and lactic acid with a depletion in level of pyruvic acid in both the tissues. This situation in the uterus indicates the condition of anti-implantation in presence of both gossypol and lactic acid. The NAD-LDH activity was inhibited in presence of gossypol and activated in presence of lactic acid in both tissues.

Effect of in vivo electrical stimulation on the carbohydrate metabolism of control and denervation atrophied muscle of dog, Canis domesticus.

The standardized programme of electrical stimulation was applied to the control and denervation atrophied muscle of dog, Canis domesticus and the pattern of changes in the carbohydrate metabolism was analysed in the control (C), denervated control (DC), control stimulated (CS) and denervated stimulated (DS) gastrocnemius muscles. The programme of electrical stimulation of the control muscle has elevated glycogenolysis, glycolysis and increased the level of operation of TCA cycle with decreased mobilization of carbohydrates into hexose monophosphate pathway, indicating the setting in of trained condition. Sciatectomy, on the other hand, lowered the level of operation of glycogenolysis and decreased the utilization of carbohydrates through hexose-mono and di-phosphate pathways and TCA cycle. The programme of electrical stimulation applied to the denervated muscle has restored the utilization of carbohydrates through hexose mono and diphosphate pathways and oxidative metabolism indicating the applicability of this programme of electrical stimulation in the treatment of muscular atrophy.

Skeletal muscle carbohydrate metabolism during short-term and prolonged in vivo electrical stimulations.

Electrical stimulations were applied to the gastrocnemius muscle of intact frog. Rana hexadactyla (Lesson) for short-term (SMS) and prolonged (PMS) periods. Short-term muscular stimulations resulted in decreased glycogenolysis and glycolysis with depleted glycogen and lactic acid contents in the muscles and increased lactic acid content in the blood. The activity levels of SDH and MDH were decreased in SMS muscles. Prolonged muscular stimulations, on the other hand, increased glycogenolysis and glycogenolysis was suggested to be due to stepped-up glycogenesis. Tissue oxidative metabolism was also correlated with regulation of activities of enzymes concerned with anaerobic pathways.

Tissue lipid profiles in orchiectomized albino rats.

Bilateral orchiectomy has been performed in adult albino rats and tissue lipid profiles were compared with the controls. Both the reproductive and non-reproductive tissues and serum accumulated lipid with suppressed lipolysis on orchiectomy. The levels of triglycerides and cholesterol were elevated in all the tissues of orchiectomized rats. The phospholipid content of epididymis was significantly depleted, while the same was elevated in non-reproductive tissues.

Muscular training through localized in vivo electrical stimulations.

In vivo electrical stimulations were applied to the gastrocnemius muscles of intact frogs for prolonged periods which lead to improved muscular efficiency and delayed onset of fatigue. The muscular strength improvement was correlated to increased tissue contractile proteins and decreased collagen content. Elevated levels of muscular fuels, mitochondrial content, oxidative metabolism in the stimulated muscles were suggested to be responsible for the delayed onset of fatigue. In view in these characteristics regarding the improvement at physical and metabolic levels, the muscles exposed to chronic electrical stimulations were termed as trained muscles. The applicability of electrical stimulations to induce the training effects into the muscles in atrophic and dystrophic conditions to avert the muscle wastings was suggested.

Pattern of skeletal muscle chemical composition during in vivo electrical stimulations.

Electrical stimulations of low voltage, frequency and short duration of time are suitable for amphibian muscle to induce localized muscular exercise in an intact animal. The pattern of changes in the muscle chemical components on chronic exposure to repeated electrical stimulations suggested the onset of regulation on muscle metabolism leading to carbohydrate sparing process and muscular hypertrophy. The applicability of this procedure to prevent the muscle wasting in atrophic and dystrophic muscles is suggested.