Data on cell viability have long been obtained from in vitro cytotoxicity assays. Today, there is a focus on markers of cell death, and the MTT cell survival assay is widely used for measuring cytotoxic potential of a compound. However, a comprehensive evaluation of cytotoxicity requires additional assays which -measure short and long-term cytotoxicity. Assays which measure the cytostatic effects of compounds are not less important, particularly for newer anticancer agents. This overview discusses the advantages and disadvantages of different non-clonogenic assays for measuring short and medium-term cytotoxicity. It also discusses clonogenic assays, which accurately measure long-term cytostatic effects of drugs and toxic agents. For certain compounds and cell types, the advent of high throughput, multiparameter, cytotoxicity assays, and gene expression assays have made it possible to predict cytotoxic potency in vivo.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Clinical Drug Trial Registry-India, www.ctri.nic.in, CTRI/2008/091/000063.

A novel L-threonine transport system is induced in Escherichia coi cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutatons in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threoni transport system is also involved in L-serine uptake and is parially shared for L-leucine transport; L-alanine, L-valine, and L-sline did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain II KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threouine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predited from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.When incubated anaerobically in tryptone yeast extract (TYE) medium, Escherichia coli synthesizes biodegradative threonine dehydratase (EC 4.2.1.16) that catalyzes the dehydration of L-threonine and L-serine to ammonia and to the corresponding a-keto acids (32). Earlier, Hobert and Datta (18) described a synthetic medium (H4) which consists of four amino acids, threonine, serine, valine, and isoleucine, plus fumarate and cyclic AMP and supports high level of enzyme production during anaerobiosis. Recently, the structural gene for the dehydratase has been cloned on a 6.3-kilobase EcoRI DNA fragment (10, 13). A variety of experiments including DNA sequence analysis, deletion studies, minicell expression, and insertion mutagenesis (13,26,27) indicated that tdcB encoding threonine dehydratase is part of the tdcABC operon that harbors two additional genes, tdcA and tdcC (Fig. 1). In addition, genetic analysis of the cloned DNA revealed that efficient expression of the operon requires the product of a regulatory gene, tdcR, situated upstream of the tdc promoter in opposite transcriptional orientation (28).The enzymatic function of the tdcB gene product in threonine degradation is well established (10); however, the physiological significance of TdcA and TdcC is not yet understood. Published reports (cited in reference 22) indicate that most catabolic pathways, especially those involved in the utilization of various amino acids and sugars, include a specific protein(s) for transport of metabolites into the cell and that some catabolic operons, such as lac and tna, harbor genes coding for their respective permeases lacY and tnaB (4, 30). For the tdc operon, both threonine and serine are required for expression of the tdc genes in the synthetic H4 medium (18), and thus far no E. coli gene specific for L-threonine and L-serine transport has been identified. These consider...

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important component of antiarthritic drugs in Ayurvedic medicine. Clinical data suggest that elevated levels of hyaluronidase and collagenase type 2 enzymes contribute significantly to cartilage degradation. Triphala guggulu (TG) is a guggulu-based formulation used for the treatment of arthritis. We assessed the chondroprotective potential of TG by examining its effects on the activities of pure hyaluronidase and collagenase type 2 enzymes. Triphala shodith guggulu (TSG), an intermediate in the production of TG, was also examined. A spectrophotometric method was used to assay Hyaluronidase activity, and to detect potential Hyaluronidase inhibitors. Aqueous and hydro-alcoholic extracts of TSG showed weak but dose-dependent inhibition of hyaluronidase activity. In contrast, the TG formulation was 50 times more potent than the TSG extract with respect to hyaluronidase inhibitory activity. A validated X-ray film-based assay was used to measure the gelatinase activity of pure collagenase type 2. Hydro-alcoholic extracts of the TG formulation were 4 times more potent than TSG with respect to collagenase inhibitory activity. Components of Triphala were also evaluated for their inhibitory activities on hyaluronidase and collagenase. This is the first report to show that the T2 component of Triphala (T.chebula) is a highly potent hyaluronidase and collagenase inhibitor. Thus, the TG formulation inhibits two major enzymes that can degrade cartilage matrix. Our study provides the first in vitro preclinical evidence of the chondroprotective properties of TG.

There is a need for effective nutraceuticals for osteoarthritis care. The fruit of Phyllanthus emblica is used as a powerful rejuvenator in Ayurvedic medicine. This study measured the chondroprotective potential of P. emblica (‘Amalaki’) fruits in vitro. We used aqueous extracts of unprocessed P. emblica fruit powder (powder A), and the powder obtained after hot water extraction and drying of powder A (powder B). Chondroprotection was measured in three different assay systems. First, we tested the effects of both fruit powders on the activities of the enzymes hyaluronidase and collagenase type 2. Second, an in vitro model of cartilage degradation was set-up with explant cultures of articular knee cartilage from osteoarthritis patients. Cartilage damage was assayed by measuring glycosaminoglycan release from explants treated with/without P. emblica fruit powders. Aqueous extracts of both fruit powders significantly inhibited the activities of hyaluronidase and collagenase type 2 in vitro. Third, in the explant model of cartilage matrix damage, extracts of glucosamine sulphate and powder B (0.05 mg/ml) exhibited statistically significant, long-term chondroprotective activity in cartilage explants from 50% of the patients tested. This result is important since glucosamine sulphate is the leading nutraceutical for osteoarthritis. Powder A induced a statistically significant, short-term chondroprotective activity in cartilage explants from all of the patients tested. This is the first study to identify and quantitate new chondroprotective activities of P. emblica fruits. These data provide pilot pre-clinical evidence for the use of P. emblica fruits as a chondroprotective agent in osteoarthritis therapy.

No abstract

This is the first report describing two novel chondroprotective activities of aqueous extracts of Withania somnifera root powder.First,these extracts had a statistically significant,short-term chondroprotective effect on damaged human osteoarthritic cartilage matrix in 50% of the patients tested. Second,these extracts caused a significant and reproducible inhibition of the gelatinase activity of collagenase type 2 enzyme in vitro.