The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.     

New furanoflavanoids,intestinal α-glucosidase inhibitory and free-radical (DPPH) scavenging,activity from antihyperglycemic root extract of Derris indica (Lam.)

A bioassay-guided fractionation and chemical examination of antihyperglycemic root extract of Derris indica resulted in isolation and characterization of two new furanoflavanoids (1, 2) along with thirteen known compounds (3–15). Their structures were determined on the basis of extensive spectroscopic (IR, MS, 1D and 2D NMR) data analysis and by comparison with the literature data. All the compounds were tested in vitro for intestinal α-glucosidase inhibitory and DPPH radical activity. New compounds (1, 2) displayed moderate intestinal α-glucosidase inhibitory as well as free radical scavenging activity. Other compounds also displayed varying degrees of moderate intestinal α-glucosidase inhibitory activity. Pongamol (6) displayed potent intestinal α-glucosidase inhibition.     

New Labdane diterpenes as intestinal α-glucosidase inhibitor from antihyperglycemic extract of Hedychium spicatum (Ham. Ex Smith) rhizomes

Phytochemical investigation of antihyperglycemic extract of rhizomes of Hedychium spicatum led to the isolation of two new labdane type diterpenes 2, 3 along with seven known compounds (1, 4–9). Their structures were established on the basis of NMR (1D and 2D) and mass spectroscopic analysis. The new compound 2 displayed strong intestinal α-glucosidase inhibitory activity. Other compounds also displayed varying degree of intestinal α-glucosidase inhibitory potential.     

Disease-specific gene repositioning in breast cancer

Genomes are nonrandomly organized within the three-dimensional space of the cell nucleus. Here, we have identified several genes whose nuclear positions are altered in human invasive breast cancer compared with normal breast tissue. The changes in positioning are gene specific and are not a reflection of genomic instability within the cancer tissue. Repositioning events are specific to cancer and do not generally occur in noncancerous breast disease. Moreover, we show that the spatial positions of genes are highly consistent between individuals. Our data indicate that cancer cells have disease-specific gene distributions. These interphase gene positioning patterns may be used to identify cancer tissues.     

Recombinant expression, purification, and biochemical characterization of chondroitinase ABC II from Proteus vulgaris

Chondroitin lyases (or chondroitinases) are a family of enzymes that depolymerize chondroitin sulfate (CS) and dermatan sulfate (DS) galactosaminoglycans, which have gained prominence as important players in central nervous system biology. Two distinct chondroitinase ABC enzymes, cABCI and cABCII, were identified in Proteus vulgaris. Recently, cABCI was cloned, recombinantly expressed, and extensively characterized structurally and biochemically. This study focuses on recombinant expression, purification, biochemical characterization, and understanding the structure-function relationship of cABCII. The biochemical parameters for optimal activity and kinetic parameters associated with processing of various CS and DS substrates were determined. The profile of products formed by action of cABCII on different substrates was compared with product profile of cABCI. A homology-based structural model of cABCII and its complexes with CS oligosaccharides was constructed. This structural model provided molecular insights into the experimentally observed differences in the product profile of cABCII as compared with that of cABCI. The critical active site residues involved in the catalytic activity of cABCII identified based on the structural model were validated using site-directed mutagenesis and kinetic characterization of the mutants. The development of such a contaminant-free cABCII enzyme provides additional tools to decode the biologically important structure-function relationship of CS and DS galactosaminoglycans and offers novel therapeutic strategies for recovery after central nervous system injury.     

Identification and evaluation of antioxidant, analgesic/anti-inflammatory activity of the most active ninhydrin-phenol adducts synthesized

Treatment of phenols with ninhydrin in acidic medium afforded 2-hydroxy-2-(ortho-hydroxy-phenyl/naphthyl)-1,3-dioxoindanes, which being unstable were isolated in their hemiketal forms. These synthesized compounds were subjected to TLC screening for radical scavenging and in vitro lipoxgenase and cycloxygenase enzyme inhibition assays. The best compound was identified and studied in detail for steady-state and time-resolved free radical kinetics, viz., DPPH, ABTS(-), *OH and rate constants for these reactions were evaluated. The best compound was also subjected to in vivo anti-inflammatory and analgesic activities in which the compound showed good promise for further structural optimization.     

Anti-tumor and proapoptotic effect of novel synthetic benzophenone analogues in Ehrlich ascites tumor cells

A series of substituted benzophenone analogues, (2-aroyl-4-methylphenoxy)acetamides 4a-e, have been synthesized via three-step synthesis sequence beginning with the 2-hydroxybenzophenones 1a-e in excellent yield. 1a-e on reaction with ethyl chloroacetate afford ethyl (2-aroyl-4-methylphenoxy)acetates 2a-e which on alkaline hydrolysis afforded (2-aroyl-4-methylphenoxy)ethanoic acid 3a-e. Compounds 3a-e on condensation with p-chloroaniline furnished benzophenone analogues 4a-e. In the present report, we investigated the anti-tumor and proapoptotic effect of benzophenones in Ehrlich ascites tumor (EAT) cells. Treatment of benzophenones in vivo resulted in inhibition of proliferation of EAT cells and ascites formation. Further, we demonstrate that the induction of apoptosis in EAT cells is mediated through activation of caspase-3. These results suggest a further possible clinical application of these synthetic compounds as potent anti-tumor and proapoptotic compounds.     

Scopolamine Induced Amnesia is Reversed by <Emphasis Type="Italic">Bacopa monniera</Emphasis> Through Participation of Kinase-CREB Pathway

Scopolamine, an anticholinergic drug, is reported to produce amnesia by interference of long term potentiation and has been used for discerning the efficacy of various antiamnesic drugs. The intoxication with anticholinergics and benzodiazepines tend to produce neurodegeneration which cause memory deficits. Our earlier reports have shown the antiamnesic drug, B. monniera to be capable of alleviating diazepam induced memory deficits. We have now tested how scopolamine affects downstream signaling molecules of long term potentiation and if B. monniera can also modulate the scopolamine induced amnesia. We used Morris water maze scale to test the amnesic effect of scopolamine and its reversal by B. monniera. Rota-rod test was used to screen muscle coordination activity of mice before water maze investigations were carried out. The results showed that scopolamine downregulated protein kinase C and iNOS without affecting cAMP, protein kinase A, calmodulin, MAP kinase, nitrite, CREB and pCREB. B. monniera reversed the scopolamine induced amnesia by significantly improving calmodulin and by partially attenuating protein kinase C and pCREB. These observations suggest involvement of calmodulin in evoking antiamnesic effects of B. monniera.     

Structural and functional characterization of HMG-COA reductase from Artemisia annua

Plants synthesize a great variety of isoprenoid products that are required not only for normal growth and development but also for their adaptive responses to environmental challenges. However, despite the remarkable diversity in the structure and function of plant isoprenoids, they all originate from a single metabolic precursor, mevalonic acid. The synthesis of mevalonic acid is catalysed by the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG- CoA reductase). The analysis of the amino acid sequence of HMG-CoA reductase from Artemisia annua L. plant showed that it belongs to class I HMG-CoA reductase family. The three dimensional structure of HMG-CoA reductase of Artemisia annua has been generated from amino acid sequence using homology modelling with backbone structure of human HMG-CoA reductase as template. The model was generated using the SWISS MODEL SERVER. The generated 3-D structure of HMG-CoA reductase was evaluated at various web interfaced servers to checks the stereo interfaced quality of the structure in terms of bonds, bond angles, dihedral angles and non-bonded atom-atom distances, structural as well as functional domains etc. The generated model was visualized using the RASMOL. Structural analysis of HMG-CoA reductase from Artemisia annua L. plant hypothesize that the N and C-terminals are positioned in cytosol by the two membrane spanning helices and the C-terminals domain shows similarity to the human HMG-CoA reductase enzyme indicating that they both had potential catalytic similarities.