A synthesis of (±)-thia-calanolide A, its resolution and in vitro biological evaluation.

A synthesis of (±)-thia-calanolide A 3 has been successfully accomplished starting from 3,5-dimethoxythiophenol 4, in six steps in an overall yield of 4.5%. The key reaction involved Friedel-Crafts tigloylation of 5,7-dihydroxy-4-n-propyl thiocoumarin 6 employing an appropriate solvent of CS2-PhNO2 in a ratio of 7:3. In its biological evaluation for anti-HIV activity, (±)-thia-calanolide A 3 demonstrated comparatively less activity with calanolide A and its synthetic analogue aza-calanolide. Further, (±)-3 has been resolved by chiral HPLC to (+) and (-)-3.

Cultured cerebellar granule neurons as an in vitro aging model: topoisomerase IIß as an additional biomarker in DNA repair and aging.

Aging in the brain is a multicellular process manifesting as neurodegeneration and associated functional impairment. In the present study, we report that cerebellar granule neurons (CGNs) in culture show senescence-mediated molecular changes indicating establishment of aging processes in vitro. CGNs were viable for 5 weeks followed by cellular degeneration. Molecular changes correlated with cellular senescence and aging include the elevation of senescence-mediated beta galactosidase (SA-ß-gal) activity and intracellular Ca(2+) levels. Decreased base excision repair (BER) as well as non-homologous end joining (NHEJ) activities in CGNs were also observed upon aging in vitro. The decrease in NHEJ activity was shown correlated with corresponding decrease in the levels of topoisomerase IIß (topo IIß), Ku 70 and Ku 80 suggesting a crucial role for topo IIß in repair capacity of CGNs. These studies, besides establishing that CGNs would serve as a good in vitro model for analysis of aging phenomena, also brought out that topo IIß, by virtue of its significant role in controlling NHEJ activity, would serve as an additional biomarker for studying aging process.

Neurotoxic activity of a topoisomerase-I inhibitor, camptothecin, in cultured cerebellar granule neurons.

DNA Topoisomerase-I (Topo-I) is an enzyme involved in DNA rearrangements, transcription and replication and is a potential target for cancer chemotherapy. Camptothecin is one of the chemotherapeutic agents inhibiting the catalytic activity of Topo-I through the formation of single-strand protein-DNA cross-links. Here, we show that camptothecin is toxic to primary cerebellar granule neurons (CGNs), and camptothecin-induced toxicity is not solely due to the inhibition of Topo-I. An analysis of the effect of camptothecin on CGNs in culture showed that camptothecin inhibits the viability of CGNs. The observed inhibition of cell viability is through the induction of a pro-apoptotic pathway that leads to neuronal degeneration. Furthermore, results show that camptothecin inhibits both protein synthesis and the neuritic outgrowth of CGNs. To determine if the observed neurotoxicity was due to inhibition of Topo-I alone, siRNA-mediated Topo-I-downregulated CGNs were analyzed for cell viability, apoptosis, protein synthesis and neurite outgrowth. The results of these experiments demonstrate that Topo-I downregulation affects only neurite outgrowth and has no significant effect on viability, apoptosis and protein synthesis in granule neurons. In conclusion, camptothecin-induced neurotoxicity may be due to the induction of protein-DNA cross-links and other unknown drug-related interactions rather than the inhibition of Topo-I activity alone.

Design, synthesis, and discovery of novel non-peptide inhibitor of Caspase-3 using ligand based and structure based virtual screening approach.

Caspase-3 belonging to a family of cysteine proteases is main executioner of apoptotic cascade pathway. The inhibitors of this protein are useful in the treatment of cardiomyopathy and neurodegenerative diseases. For the discovery of novel Caspase-3 non-peptide inhibitors from Maybridge database, ligand based and structure based virtual screening methods were used. Quantitative 3D pharmacophore models were generated using 25 known inhibitors of Caspase-3 and it was used as initial screen to retrieve the hits from the database. These compounds with high estimated activity were analyzed for drug like properties and docking studies were performed, to study the interaction between new hits and active site. One of the hits (AW01208), with good predictions was selected for synthesis and biological screening. This compound showed an inhibition activity against Caspase-3 in SKNH cell lines.

Biocompatibility of adenoviral vectors in poly(vinyl chloride) tubing catheters with presence or absence of plasticizer di-2-ethylhexyl phthalate.

Adenoviral (Ad) vectors feature attractive characteristics for gene therapy of a wide variety of diseases. In many cases, the Ad vector must be administered using catheters and other plastic medical devices. Although poly(vinyl chloride) is one of the most frequently used catheter materials, it is relatively rigid and requires the addition of a plasticizer such as di-2-ethylhexyl phthalate (DEHP) to increase its flexibility. In this study, we demonstrated that exposure to a DEHP-containing catheter decreased the infectivity of Ad vectors but not the total particle number of the vector. Loss of Ad vector infectivity was directly related to the time of exposure to the DEHP-containing catheter, but it was not due to simple leaching of the chemical from the plastic. The loss of Ad vector infectivity could be prevented by preflushing the tube with albumin. Careful consideration of the compatibility between gene therapy vectors and medical delivery devices will be critical to the success of human gene therapy applications.

Metabolic and cardiac effects of Clistanthus collinus poisoning.

A prospective study of 11 cases of Clistanthus collinus leaf poisoning was undertaken to evaluate the mechanism of hypokalaemia that occurs in these cases. Continuous cardiac monitoring was done. Ventricular ectopics, ventricular tachycardia and fibrillation were the important arrhythmias. No atrioventricular blocks or other significant bradyarrhythmias were noted. The renal potassium loss was found to be very high (120 +/- 87.95 mEq/L) even in the presence of hypokalaemia and in spite of continuing drop in serum potassium concentration. Metabolic study established that renal potassium leak is the mechanism by which hypokalaemia was mediated.