An in silico,in vitro and in vivo investigation of indole-3-carboxaldehyde identified from the seawater bacterium Marinomonas sp. as an anti-biofilm agent against Vibrio cholerae O1

Biofilm formation is a major contributing factor in the pathogenesis of Vibrio cholerae O1 (VCO1) and therefore preventing biofilm formation could be an effective alternative strategy for controlling cholera. The present study was designed to explore seawater bacteria as a source of anti-biofilm agents against VCO1. Indole-3-carboxaldehyde (I3C) was identified as an active principle component in Marinomonas sp., which efficiently inhibited biofilm formation by VCO1 without any selection pressure. Furthermore, I3C applications also resulted in considerable collapsing of preformed pellicles. Real-time PCR studies revealed the down-regulation of virulence gene expression by modulation of the quorum-sensing pathway and enhancement of protease production, which was further confirmed by phenotypic assays. Furthermore, I3C increased the survival rate of Caenorhabditis elegans when infected with VCO1 by significantly reducing in vivo biofilm formation, which was corroborated by a survivability assay. Thus, this study revealed, for the first time, the potential of I3C as an anti-biofilm agent against VCO1.     

Purification and kinetic characterization of the liverwort Pallavicinia lyelli (Hook.) S. Gray. cytosolic ascorbate peroxidase

Ascorbate peroxidase (APX) of the liverwort Pallavicinia lyelli was extracted and purified through ammonium sulfate precipitation, Butyl-Toyopearl, DEAE-Cellulofine and Sephadex G-75 chromatography. The purification factor for APX was 285 with 7.9% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of APX was approximately 28 kDa estimated by SDS-PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 6.0. The enzyme had a temperature optimum at 40 °C and was relatively stable at 60 °C, with 54% loss of activity. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity and the capacity to utilize alternate electron donors. p-chloromercuribenzoate (pCMB), hydroxyurea and salicylic acid (SA) significantly inhibited APX activity. Ascorbate (AsA) and pyrogallol were found to be efficient substrates for Pallavicinia APX, considering the Vmax/Km ratio. We detected the activity of monodehydroascorbate reductase (MDHAR) involved in the regeneration of ascorbate, but failed to detect the dehydroascorbate reductase (DHAR) activity. The data obtained in this study may help to understand desiccation tolerance mechanism in the liverwort.     

2′,3′-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening

The recent report of 2′,3′-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2′,3′-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2′,3′-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3′-AMP but not 2′-AMP. The catalytic efficiency (k_cat/K_m) of each enzyme against 2′,3′-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2′,3′-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3′-AMP is due to the P-O2′ bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2′,3′-cAMP hydrolysis is observed.     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.     

Testing the reliability of genetic methods of species identification via simulation

Although genetic methods of species identification, especially DNA barcoding, are strongly debated, tests of these methods have been restricted to a few empirical cases for pragmatic reasons. Here we use simulation to test the performance of methods based on sequence comparison (BLAST and genetic distance) and tree topology over a wide range of evolutionary scenarios. Sequences were simulated on a range of gene trees spanning almost three orders of magnitude in tree depth and in coalescent depth; that is, deep or shallow trees with deep or shallow coalescences. When the query's conspecific sequences were included in the reference alignment, the rate of positive identification was related to the degree to which different species were genetically differentiated. The BLAST, distance, and liberal tree-based methods returned higher rates of correct identification than did the strict tree-based requirement that the query was within, but not sister to, a single-species clade. Under this more conservative approach, ambiguous outcomes occurred in inverse proportion to the number of reference sequences per species. When the query's conspecific sequences were not in the reference alignment, only the strict tree-based approach was relatively immune to making false-positive identifications. Thresholds affected the rates at which false-positive identifications were made when the query's species was unrepresented in the reference alignment but did not otherwise influence outcomes. A conservative approach using the strict tree-based method should be used initially in large-scale identification systems, with effort made to maximize sequence sampling within species. Once the genetic variation within a taxonomic group is well characterized and the taxonomy resolved, then the choice of method used should be dictated by considerations of computational efficiency. The requirement for extensive genetic sampling may render these techniques inappropriate in some circumstances.     

Taxol Determination from Pestalotiopsis pauciseta, a Fungal Endophyte of a Medicinal Plant

Taxol is the most effective antitumor agent developed in the past three decades. It has been used for effective treatment of a variety of cancers. A taxol-producing endophytic fungus Pestalotiopsis pauciseta (strain CHP-11) was isolated from the leaves of Cardiospermum helicacabum and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores and screened for taxol production. The amount of taxol produced by this endophytic fungus was quantified by HPLC and it produced 113.3 mg/L, thus the fungus can serve as a potential material for fungus engineering to improve taxol production. This fungal taxol also had strong anticancer activity against some cancer cells viz., BT 220, H116, Int 407, HL 251 and HLK 210 tested by Apoptotic assay and it is indicated that with the increase of taxol concentration from 0.005–0.05 mmol/L, taxol induced increased cell death through apoptosis.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

The polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution.