Semi-synthetic aristolactams—inhibitors of CDK2 enzyme

Several analogs of aristolochic acids were isolated and derivatized into their lactam derivatives to study their inhibition in CDK2 assay. The study helped to derive some conclusions about the structure–activity relation around the phenanthrin moiety. Semi-synthetic aristolactam 21 showed good activity with inhibition IC₅₀ of 35 nM in CDK2 assay. The activity of this compound was comparable to some of the most potent synthetic compounds reported in the literature.     

The crystal structure of a TIR domain from Arabidopsis thaliana reveals a conserved helical region unique to plants

Plants use a highly evolved immune system to exhibit defense response against microbial infections. The plant TIR domain, together with the nucleotide‐binding (NB) domain and/or a LRR region, forms a type of molecule, named resistance (R) proteins, that interact with microbial effector proteins and elicit hypersensitive responses against infection. Here, we report the first crystal structure of a plant TIR domain from Arabidopsis thaliana (AtTIR) solved at a resolution of 2.0 Å. The structure consists of five β‐strands forming a parallel β‐sheet at the core of the protein. The β‐strands are connected by a series of α‐helices and the overall fold mimics closely that of other mammalian and bacterial TIR domains. However, the region of the αD‐helix reveals significant differences when compared with other TIR structures, especially the αD3‐helix that corresponds to an insertion only present in plant TIR domains. Available mutagenesis data suggest that several conserved and exposed residues in this region are involved in the plant TIR signaling function.     

A liquid chromatography-tandem mass spectrometric method for quantification of curcuminoids in cell medium and mouse plasma

Curcumin and tetrahydrocurcumin (THC) have been found as potent DNMT1 inhibitors, but they suffer from low oral bioavailability and rapid metabolism in vivo. To circumvent these problems, two curcumin analogs: 1,7-bis(3,4-dimethoxyphenyl)-4,4-dimethyl-1,6-heptadiene-3,5-dione (TMC) and 1,7-bis(3,4-dimethoxyphenyl)-4-cyclohexyl-1,6-heptadiene-3,5-dione (DMCHC) have been synthesized to enhance their stability by blocking the two metabolic sites, the phenolic and C4 methylene moieties. Both compounds have shown inhibitory activity on M. SssI similar to that of curcumin and THC (Poster, M1114, AAPS, 2009). Preclinical pharmacokinetics has yet to be performed. In this paper, a simple liquid chromatography-tandem mass spectrometric method was developed for the determination of these four curcuminoids in cell medium and mouse plasma. The method showed linearity from 1 to 1000 ng/mL with the lower limit of quantification of 1 ng/mL in cell medium, and 5 ng/mL in mouse plasma for all test curcuminoids. The within-day coefficients of variation were found to be below 15% and the accuracy was in the range of 85-115%. This method was subsequently used to evaluate their stability in these matrices and a pilot pharmacokinetics of curcumin, DMCHC and TMC in mice after an intraperitoneal (i.p.) cassette dosing of 10mg/kg each. Curcuminoids degraded in two phases with terminal half lives of 186, 813, 724, and 2000 min for curcumin, THC, TMC, and DMCHC, respectively, in cell culture medium. In plasma, their respective half lives were 111, 232, 1202 and 3000 min. These data demonstrated that their stability is in the order curcumin相似文献   

Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C₄-HSL (rhlR–rhlI-dependent QS) and 3-oxo-C₁₂-HSL (lasR–lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C₁₂-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

Presently there is no serum biomarker of rhabdomyosarcoma (RMS). Several studies have shown that profiles of microRNA (miRNA) expression differ among tumor types. Here we evaluated the feasibility of using muscle-specific miRNAs (miR-1, -133a, -133b and -206) as biomarkers of RMS. Expression of muscle-specific miRNAs, especially miR-206, was significantly higher in RMS cell lines than in other tumor cell lines, as well as in RMS tumor specimens. Further, serum levels of muscle-specific miRNAs were significantly higher in patients with RMS tumors than in patients with non-RMS tumors. Normalized serum miR-206 expression level could be used to differentiate between RMS and non-RMS tumors, with sensitivity of 1.0 and specificity of 0.913. These results raise the possibility of using circulating muscle-specific miRNAs, especially miR-206, as landmark biomarkers for RMS.