A high-throughput screening assay for simultaneous selection of inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) or 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr)

1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis (Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (ΔH -10.2 kcal/mol, ΔS 1.1 cal mol(-1)K(-1)). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an HTS assay that couples Dxr to the upstream enzyme 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), also a valid target, was developed. A high concentration of NADPH was used to bias it to detect Dxr inhibitors that bind like fosmidomycin. The assay Z' was 0.75. It was equally sensitive to inhibitors of Dxs and Dxr, that is, fosmidomycin and fluropyruvate inhibited it with IC(50)s similar to that in the individual enzyme assays (79 vs 54 nM for fosmidomycin). To distinguish inhibitors of Dxs from Dxr, individual enzyme assays and a microplate thermofluor binding assay were developed. The assay simultaneously screens two targets and is cost-effective.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Computational prediction of residues involved in fidelity checking for DNA synthesis in DNA polymerase I

Recent single-molecule F?rster resonance energy transfer studies of DNA polymerase I have led to the proposal of a postinsertion fidelity-checking site. This site is hypothesized to ensure proper base pairing of the newly inserted nucleotide. To help test this hypothesis, we have used energy decomposition, electrostatic free energy response, and noncovalent interaction analysis analyses to identify residues involved in this putative checking site. We have used structures of DNA polymerase I from two different organisms, the Klenow fragment from Escherichia coli and the Bacillus fragment from Bacillus stearothermophilus. Our results point to several residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Furthermore, many of these residues are conserved among A family polymerases. The identified residues provide potential targets for mutagenesis studies for investigation of the fidelity-checking site hypothesis.     

Tamarindus indica L. leaf is a source of allelopathic substance

The allelopathic potential of the Tamarindus indica L. leaf was investigated through bioassay guided studies using several weed and edible crop species. Both radicle and hypocotyl growth of all the plant species tested was strongly inhibited by the tamarind leaf using a sandwich method. The growth of weed species was reduced more than that of edible crop species. Among the weed species, barnyard grass followed by white clover, and in the edible crop species, lettuce followed by radish ranked top in terms of growth inhibition. Different concentrations of tamarind leaf crude water-soluble extract exhibited a strong inhibition in all the plant species tested and, by contrast, the magnitude of inhibition in the weed species was higher than in edible crop species and ranged from 30–75%. The 10% concentration of the tamarind leaf crude water-soluble extract was most potent against growth of seedlings. The concentrations of the nutrient components were linearly correlated with an increase in the concentration of tamarind leaf crude water-soluble extract. No significant changes in either pH or EC were found in the variations of different concentrations of tamarind leaf crude water-soluble extracts. As compared to control, growth of both radicle and hypocotyl in weed (barnyard grass and white clover) and in edible crop (lettuce and radish) species were significantly reduced when blended tamarind leaves at different concentrations were incorporated into the growth medium. The inhibitory magnitude increased with an increase in the concentration of the tamarind leaf. In terms of growth inhibition, among these tested plants, weed species particularly barnyard grass were most sensitive to the allelochemicals exuded from blended tamarind leaves. When the blended tamarind leaves were removed from the growth medium, all the seedlings grew quickly and the percentage of recovery was between 76–97% of the corresponding controls. Reduction in the fresh and dry weight of these 4 plant species was observed under the experimental conditions, and ranged between 33–42% and 40–53% in the radicle and hypocotyl, respectively. The fresh and dry weight, and total chlorophyll content declined significantly in the incorporated tamarind leaf treatments. Compared to the control, the highest drop in the chlorophyll content of 60% in barnyard grass was observed with the 10% concentration of the leaf treatment. These results clearly indicate that the tamarind leaf contains one or more strong biologically active allelochemical(s) that function as true growth regulator(s) and is involved in plant growth regulation, particularly in weed species.     

Alpha(v)beta(6) integrin-A marker for the malignant potential of epithelial ovarian cancer.

The mechanism(s) responsible for the progression of non-metastatic or borderline ovarian cancer to invasive Grade I/III ovarian cancer is still unknown. An epithelium-restricted integrin, alpha(v)beta(6), is present in malignant epithelia but not in normal epithelia. We studied the relative expression and distribution of alpha(v)beta(6) integrin in early and late-stage invasive (Grade I and Grade III) and non-invasive (benign and borderline) ovarian tumors of serous, mucinous, endometrioid, and clear-cell carcinoma subtypes, to assess its potential as a marker for epithelial ovarian cancer progression. Sixty-six specimens, including eight normal, 13 benign, 14 borderline, 13 Grade I, and 18 Grade III tumors were evaluated by immunohistochemistry (IHC) using a monoclonal antibody (MAb) against alpha(v)beta(6) integrin. Normal ovarian surface epithelium was negative for alpha(v)beta(6) integrin expression. All 45 carcinomas studied were positive, and the staining intensity significantly correlated with the grade of the tumor. The Grade III carcinomas of all types showed strong staining intensity. Only mucinous benign tissues were positive, and no reactivity was observed in benign serous neoplasms. On the basis of these observations, we hypothesize that the expression of alpha(v)beta(6) integrin is associated with epithelial ovarian cancer and that a gradual increase in the expression of the molecule may be a correlative index of the progression of this disease.     

DNA photolyase of enterococci: possible explanation for its low sunlight inactivation rate

DNA photolyase is perhaps the most ancient and direct arsenal in curing the UV-induced dimers formed in the microbial genome. Out of two cofactors of the enzyme, catalytic and light harvesting, differences in the latter have provided basis for categorizing photolyases of prokaryotes as folate and deazaflavin types. In the present study, the homology modeling of DNA photolyase of Enterococcus faecalis was undertaken. The predicted models were structurally compared with the crystal structure coordinates of photolyases from Escherichia coli (folate type) and Anacystis nidulans (deazaflavin type). Discrepancies present in the multiple sequence alignment and tertiary structures, particularly at the light harvesting cofactor (methenyltetrahydrofolic acid, MTHF; 8-hydroxy-5-deazaflavin, 8-HDF) binding sites indicated the mechanistic nature of enterococcal photolyase. Concisely, despite the greater holistic homology with folate-type photolyase, enterococcal photolyase was characterized as deazaflavin-type. The presence of 8-HDF binding sites and groove architecture of substrate binding sites were also found supportive in this regard. The inter cofactor distance and/or orientation also implied to the efficient energy transfer in photolyase of Enterococcus in comparison with E. coli. In addition, we observed relatively high protein deformability in the enterococcal genome, which may favors the repair action of photolyase. The findings are expected to provide molecular insights into the difference in sunlight inactivation rate of two important fecal contamination indicators, namely Enterococcus and E. coli.     

Role of inositol polyphosphates in programed cell death in Dictyostelium discoideum and its developmental life cycle

Molecular and Cellular Biochemistry - Carvacrol is a natural phenolic compound found in essential oils of Lamiaceae species. In the present study, an attempt has been made to elucidate the...     

Genome analysis of amaranths: Determination of inter- and intra-species variations

Amaranths are an important group of plants and include grain, vegetable and ornamental types. Despite the economic importance of the amaranths, there is very little information available about the extent and nature of genetic diversity present in the genus Amaranthus at molecular level. We now report the randomly amplified polymorphic DNA (RAPD) profiles of different species of Amaranthus as well as different accessions of the species. These RAPD analyses have been carried out using 65 arbitrary sequence decamer primers. From the RAPD data, an UPGMA dendrogram illustrating the inter-as well as intra-species relationships has been computed. The putative hybrid origin of A.dubious from A. hybridus and A. spinosus is also ruled out by the RAPD data. The trends of species relationships amongst the amaranths determined by RAPDs is consistent with their cytogenetic and evolutionary relationships that have already been determined. NBRI Communication No:464 (N.S.).