Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

A robust two-way semi-linear model for normalization of cDNA microarray data

Background  

Normalization is a basic step in microarray data analysis. A proper normalization procedure ensures that the intensity ratios provide meaningful measures of relative expression values.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

Re-evaluation of the function of the male specific lethal complex in Drosophila

A set of proteins and noncoding RNAs,referred to as the male specific lethal (MSL) complex,is present on the male X chromosome in　Drosophila and has been postulated to be responsible for dosage compensation of this chromosome - the up-regulation of its expression to be　equal to that of two X chromosomes in females.This hypothesis is evaluated in view of lesser known aspects of dosage compensation such as the　fact that metafemales with three X chromosomes also have equal expression to normal females,which would require a down-regulation of each　gene copy.Moreover,when this complex is ectopically expressed in females or specifically targeted to a reporter in males,there is no increase in　expression of the genes or targets with which it is associated.These observations are not consistent with the hypothesis that the MSL complex　conditions dosage compensation.A synthesis is described that can account for these observations.     

Genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.     

Biodegradable amphiphilic triblock copolymer bearing pendant glucose residues: preparation and specific interaction with Concanavalin A molecules

A novel biodegradable amphiphilic block copolymer PLGG-PEG-PLGG bearing pendant glucose residues is successfully prepared by the coupling reaction of 3-(2-aminoethylthio)propyl-alpha-D-glucopyranoside with the pendant carboxyl groups of PLGG-PEG-PLGG in the presence of N,N'-carbonyldiimidazole. The polymer PLGG-PEG-PLGG, i.e., poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-block-poly(ethylene glycol)-block- poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}, is prepared by ring-opening copolymerization of L-lactide (LLA) with (3s)-benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 2000 as macroinitiator and Sn(Oct)2 as catalyst, and then by catalytic hydrogenation. The glucose-grafted copolymer shows a lower degree of cytotoxicity to ECV-304 cells and improved specific recognition and binding with Concanavalin A (Con A). Therefore, this kind of glucose-grafted copolymer may find biomedical applications.     

3D QSAR studies on peroxisome proliferator-activated receptor γ agonists using CoMFA and CoMSIA

The peroxisome proliferator-activated receptors (PPARs) have increasingly become attractive targets for developing novel anti-type 2 diabetic drugs. We employed comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) to study three-dimensional quantitative structure–activity relationship (3D QSAR) based on existing agonists of PPAR (including five thiazolidinediones and 74 tyrosine-based compounds). Predictive 3D QSAR models with conventional r² and cross-validated coefficient (q²) values up to 0.974 and 0.642 for CoMFA and 0.979 and 0.686 for COMSIA were established using the SYBYL package. These models were validated by a test set containing 18 compounds. The CoMFA and CoMSIA field distributions are in general agreement with the structural characteristics of the binding pockets of PPAR, which demonstrates that the 3D QSAR models built here are very useful in predicting activities of novel compounds for activating PPAR.