Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor

Blue light induces carotenogenesis in Myxococcus xanthus. The carB operon encodes all but one of the structural genes involved, and its expression is regulated by the CarA-CarS repressor-antirepressor pair. In the dark, CarA-operator binding represses carB. CarS, produced on illumination, interacts physically with CarA to dismantle the CarA-operator complex and activate carB. Both operator and CarS bind to the autonomously folded N-terminal domain of CarA, CarA(Nter), which in excess represses carB. Here, we report the NMR structure of CarA(Nter), and map residues that interact with operator and CarS by NMR chemical shift perturbations, and in vivo and in vitro analyses of site-directed mutants. We show CarA(Nter) adopts the winged-helix topology of MerR-family DNA-binding domains, and conserves the majority of the helix-turn-helix and wing contacts with DNA. Tellingly, helix alpha2 in CarA, a key element in operator DNA recognition, is also critical for interaction with CarS, implying that the CarA-CarS protein-protein and the CarA-operator protein-DNA interfaces overlap. Thus, binding of CarA to operator and to antirepressor are mutually exclusive, and CarA may discern structural features in the acidic CarS protein that resemble operator DNA. Repressor inactivation by occluding the DNA-binding region may be a recurrent mechanism of action for acidic antirepressors.     

Influence of process conditions on end product formation from Clostridium thermocellum 27405 in solid substrate cultivation on paper pulp sludge

Solid substrate cultivation of thermophilic, anaerobic bacteria offers an alternative production method for many bio-based chemicals; however the process must be optimized for each substrate-organism fermentation. The effects of initial substrate moisture content (SMC, 30%, 50% and 70% wet-basis), supplemental nutrient concentration (SNC, 12%, 50% and 100%) and duration of cultivation time (6, 10 and 14 days), on product formation (lactate, ethanol and acetate) by Clostridium thermocellum 27405 were examined during growth on paper pulp sludge. Water activities at moisture contents above 30% wet-basis were essentially identical ( approximately 0.99), yet the water contents differed significantly, and affected the metabolic activity of C. thermocellum. Increases in initial substrate moisture content from 50% to 70% for cultures supplemented with 50% or 100% nutrients resulted in a 75-145 mM increase in total end products. At 70% SMC, the addition of 100% SNC generated a 56% increase in product formation above the addition of 50% nutrient supplementation. Increases in the quantity of free water present in the solid substrate cultivation system up to the water holding capacity of the paper pulp sludge led to improved performance of this anaerobic bacterium. While nutrient supplementation is common in the form of salts for many aerobic microorganisms, efficient metabolism for anaerobic C. thermocellum grown in SSC was highly dependent on added salts, vitamins and reducing agents. Further studies are needed to determine if this is a general effect for other anaerobes grown in solid substrate cultures.     

Synthesis and evaluation of substituted benzoisoquinolinones as potent inhibitors of Chk1 kinase

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.     

Design and synthesis of conformationally constrained tri-substituted ureas as potent antagonists of the human glucagon receptor

A series of conformationally constrained tri-substituted ureas were synthesized, and their potential as glucagon receptor antagonists was evaluated. This effort resulted in the identification of compound 4a, which had a binding IC50 of 4.0 nM and was shown to reduce blood glucose levels at 3 mg/kg in glucagon-challenged mice containing a humanized glucagon receptor. Compound 4a was efficacious in correcting hyperglycemia induced by a high fat diet in transgenic mice at an oral dose as low as 3 mg/kg.     

Differential gene expression profiles associated with heavy metal tolerance in the soil insect Orchesella cincta

Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola, springtails) populations occurring at metal-contaminated mining sites. Metal tolerance in O. cincta is correlated with heritable increase of excretion efficiency, decrease in cadmium-induced growth reduction, and over-expression of the single copy metallothionein (mt) gene. Although mt plays a major role in cadmium (Cd) detoxification an additional number of gene products may be altered in the tolerant phenotype that influence quantitative traits like excretion efficiency and growth. Using suppression subtractive hybridization analysis (SSH) genes were cloned that showed up- or down-regulation in response to Cd. This study reports about 192 SSH derived cDNA clones, of which 49 were confirmed to be differentially expressed after spot blot analysis. After sequence analysis 19 of the 49 differential clones could be annotated. Real-time RT-PCR of a reference culture and tolerant populations revealed significant gene responses upon Cd exposure. The reference culture showed stress induced gene expression, whereas the tolerant animals maintained normal gene expression or down regulation of cDNAs upon Cd exposure. Seven cDNAs with homology to genes involved in cell signaling, transport and programmed cell death showed an interaction between treatment and population.     

Screening of a novel octamer peptide, CNSCWSKD, that induces caspase-dependent cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis to various tumor cells but not in normal cells. We have screened cell death-inducing peptides from the extracellular domain sequence of TRAIL, using a peptide array. Peptides of higher activity were found through amino acid substitution, and the CNSCWSKD peptide induced >90% cell death in treated Jurkat cells. Features of apoptosis, such as DNA fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with TRAIL for binding to the death receptor (DR) 4 or DR5 were observed, suggesting that this peptide is a TRAIL mimic. Caspase-3 activation was observed in various tumor cells treated with this peptide as well as with TRAIL, while no activation was observed in human normal fibroblasts. The CNSCWSKD peptide is a potential candidate for use in cancer therapy.     

The 67kDa laminin receptor as a primary determinant of anti-allergic effects of O-methylated EGCG

Previously we have reported that the O-methylated derivative of (−)-epigallocatechin-3-O-gallate (EGCG), (−)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me), possesses anti-allergic activities such as inhibition of histamine release and suppression of the high-affinity IgE receptor (FcεRI) expression. However, the underlying mechanism is still unclear. Recently we have identified the 67 kDa laminin receptor (67LR) as a cell-surface receptor that can mediate biological activities of EGCG. Here we show that the suppression of myosin II regulatory light chain (MRLC) phosphorylation through the cell-surface binding to the 67 LR contributes to the inhibitory effect of EGCG3”Me on the histamine release from the human basophilic KU812 cells. The 67LR also mediated the EGCG3”Me-induced suppression of FcεRI expression by reducing ERK1/2 phosphorylation. These results suggest that anti-allergic effects of EGCG3”Me may be triggered by the inhibition of MRLC or ERK1/2 phosphorylation mediated through the cell-surface 67LR.