Optimization of luminescent assay for screening of cyclin-dependent kinase 2 inhibitors.

Cyclin-dependent kinases are most extensively studied targets for cancer chemotherapy since the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised. Cyclin-dependent kinases ensure the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Deregulation of the cyclin-dependent kinase 2 activity by cellular and external factors leads to many diseases like cancers. Different methods like radiolabeled, fluorescence and luminescence are available for screening of library of compounds against kinases. However, bioluminescent methods offer several advantages like low background and no effect of fluorescent compound interference. Present study is focused on development, optimization and validation of cyclin-dependent kinase 2 assay which is suitable for identification potent and selective, ATP competitive and non-competitive inhibitors of cyclin-dependent kinase 2. The aim of present investigation was to optimize the assay for cyclin-dependent kinase 2/cylin A and cyclin-dependent kinase 2/cyclin E with use of bioluminescence based biochemical reaction. Both cyclin-dependent kinase 2 which are cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E complexes, have different affinity for ATP. Therefore, both isoform analogs of cyclin-dependent kinase 2 were optimized separately. Optimum cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E concentration were found to be 250 ng/well and 200 ng/well, respectively. Optimum substrate (histone H1) concentration was found to be 2.5 mg/ml for both cyclin-dependent kinase 2 analogs. Optimum reaction time was found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes.

Antioxidant and Antidiabetic Activity of Helicteres isora (L.) Fruits.

The present investigations evaluated the antioxidant and antidiabetic activity of Helicteres isora (L.) fruits belonging to the family Sterculiaceae. The hot water extract of Helicteres isora fruits was prepared and screened for its in vitro antioxidant activity using 1,1-diphenyl,2-picryl hydrazyl assay, ss-carotene-linoleate model and microsomal lipid peroxidation or thiobarbituric acid reactive species assays and the IC(50) values were calculated. Antidiabetic effect was studied using the in vitro glucose uptake in the isolated rat hemi-diaphragm model. The hot water extract of Helicteres isora showed maximum activity with IC(50) value 25.12+/-0.18 mug/ml for 1,1-diphenyl,2-picryl hydrazyl assay method, and low activity with IC(50) value 740.64+/-4.76 mug/ml for microsomal lipid peroxidation assay. In the ss-carotene-linoleate model, the extract showed 45.63% antioxidant activity. The extract produce a significant (P<0.05) uptake of glucose by isolated rat hemi-diaphragm but less effective to that of the reference drug, metformin. The hot water extract of fruit of Helicteres isora exhibited significant antioxidant activity and moderate antidiabetic activity and merits further investigation in animal models and isolation of its active constituents.

Role of ascorbic acid on mercuric chloride-induced genotoxicity in human blood cultures.

Efforts are made to find therapeutic agents capable of minimizing genotoxicity of various natural and man-made compounds. The genotoxicity induced by mercury compounds remains controversial. Therefore we have investigated the genotoxic effect of mercuric chloride (MC; HgCl(2)) at three concentrations (1.052, 5.262 and 10.524 microM) and role of L-ascorbic acid (vitamin C) at a concentration of 9.734 microM on MC-treated short-term human leucocyte cultures. We assessed the proliferative rate index (PRI), sister chromatid exchange (SCE) and chromosomal aberrations (CAS) in control and MC-treated cultures with and without vitamin C supplementation. The results showed that MC has no effect on cell-cycle kinetics, but the frequency of SCE/cell was significantly higher in a dose-dependent manner than control values. HgCl(2) also significantly induced C-anaphases (abnormal mitosis) in blood cultures. These effects were prevented by the addition of vitamin C to MC-treated cultures. The data indicate the mutagenic activity of MC and the protective role of vitamin C on mercury-induced genotoxicity in human blood cultures is probably due to its strong antioxidant and nucleophilic nature.

Morton neuroma: evaluation with MR imaging performed with contrast enhancement and fat suppression.

PURPOSE: To evaluate clinically suspected Morton neuroma with contrast material-enhanced magnetic resonance (MR) images. MATERIALS AND METHODS: Fifteen patients with clinically suspected Morton neuroma underwent examination with conventional T1- and T2-weighted MR imaging and a combination of fat suppression and administration of gadopentetate dimeglumine. A T1-weighted spectral presaturation with inversion recovery sequence was used for fat suppression. RESULTS: In six patients, a tumor that conformed to the clinical findings was seen in the interdigital space; surgical findings in these patients correlated closely with the imaging findings in all patients. Patients without positive findings on MR images tended to have less typical clinical findings and received nonsurgical treatment. In all patients, the lesions were best depicted with the combination of contrast-enhanced imaging and fat suppression; conventional MR images either entirely failed to demonstrate the lesions or demonstrated the lesions less clearly. CONCLUSION: In patients who need imaging confirmation of a clinically suspected Morton neuroma, the combination of fat suppression and contrast enhancement provides reliable high-contrast images.

Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.

In situ hybridization was used to determine the morphologic distribution of tropoelastin and alpha 1(I) procollagen mRNA expression in elastic intralobar arteries from neonatal calves with hypoxic pulmonary hypertension induced by a 15-day exposure to a simulated altitude of 1500 m. In vessels from normotensive control animals, low levels of hybridizable tropoelastin mRNA were detected in smooth muscle cells (SMC) of the inner media associated with large elastic lamellae. Compared to control arteries, vessels from hypertensive animals demonstrated a markedly different pattern of hybridization. In these arteries, strong hybridization signals for tropoelastin mRNA were seen in SMC lying between the elastic lamellae of the outer media, and the density of labeling associated with these medial cells decreased progressively toward the lumen. Endothelial and adventitial cells in both control and hypertensive arteries were negative for tropoelastin mRNA. Type I procollagen mRNA was dispersed through the media of control arteries, and in hypertensive calves, the hybridization signal was more intense and was unevenly distributed through the media similarly to that for tropoelastin mRNA. Adventitial cells were strongly positive for procollagen mRNA, and the signal was equally intense for both control and hypertensive arteries. Cells that had no detectable tropoelastin mRNA were noted in the outer media of both control and hypertensive vessels. These cells occurred as broad circumferential bands in the normotensive artery and as nodular foci in the hypertensive artery. Immunocytochemical studies with antibodies to smooth muscle specific actin, desmin, and vimentin demonstrated that cells within these foci, as well as tropoelastin mRNA-positive cells, were SMC. These studies demonstrate that expression of tropoelastin and procollagen mRNA was differentially stimulated by pulmonary hypertension within specific regions and SMC populations of the vascular wall.