Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.     

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Analysis of gene expression profile in p130(Cas)-deficient fibroblasts

p130(Cas) (Cas) is a docking protein that becomes tyrosine phosphorylated in v-Src- or v-Crk-transformed cells and in integrin-stimulated cells. Cas -/- fibroblasts show defects in stress fiber formation, cell spreading, cell migration, and transformation by activated Src. To further characterize the role of Cas in signaling, we compared the expression profile in Cas -/- fibroblasts with that in Cas-re-expressing fibroblasts using the microarray methods. In Cas -/- fibroblasts, the expression of heme oxygenase 1 and caveolin-1 was reduced, but the expression of procollagen 1 alpha 1, procollagen 3 alpha 1, procollagen 11 alpha 1, elastin, periostin, TSC-36, and MARCKS was enhanced. The domains in Cas necessary for the change varied among these genes. Activated Src reduced the expression of most of these genes both in Cas -/- and in Cas +/+ fibroblasts. These results suggest the existence of signaling pathways that emanate from Cas to gene expression.     

Production of (R)-1-phenylethanol through enantioselective oxidation of (S)-isomer from their racemic mixture by Pachysolen tannophilus IFO 1007

Summary Stereoselective oxidation of (S)-isomer of rac-1-phenylethanol (1-PA) by the yeast Pachysolen tannophilus IFO 1007 immobilized into calcium-alginate gels was investigated to produce (R)-isomer. Continuous production of (R)-isomer was accomplished for more than 80 h with an enantiomeric excess of > 90% using a bioreactor of a fluidized-bed type.     

Development of saxitoxin-conjugated affinity gels

Saxitoxin (STX) and its analogues accumulated in bivalves cause food poisoning through the blockade of sodium channels in the nervous system. In the current studies, STX-conjugated agarose gels as affinity chromatography reagents were prepared for investigation of the fate of the toxins in natural environments and in the human body. A carboxyl moiety was introduced through positions C11 and C13 to leave the most characteristic part of the molecule intact. Two types of synthesized derivatives, 11-(2-carboxyethylthio)saxitoxin and 13-O-hemisuccinyldecarbamoylsaxitoxin, were successfully conjugated to Sepharose 4B in high yield. Affinity gels containing 500 nmol of STX or decarbamoylsaxitoxin per milliliter of gel were accomplished by masking the residual amino groups by acetylation. Finally, the STX-conjugated affinity gel was effective for concentrating STX-binding proteins from pufferfish and bullfrog plasma.     

Effects of a novel Y5 antagonist in obese mice: combination with food restriction or sibutramine

Objective: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice. Methods and Procedures: Male C57BL/6 or Npy5r^−/− mice were adapted to high-fat (HF) diet for 6–10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r^−/− mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified. Results: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r^−/− DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment. Discussion: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.     

The Influence of Measurement Parameters on the Specific Rotation of Amino Acids

The effects of solute and hydrochloric acid concentrations on optical rotation were studied using 20 naturally occuring amino acids.

There appeared to be no common factor among the amino acids as far as the inclination of optical rotation was concerned. Lutz-Jirgenson’s rule could be applied to few amino acids in the cationic form. Therefore, in the determination of the optical rotation, the concentration of the solute, nature of solvent and temperature must be rigorously controlled. The optical conditions of measurement and the specific rotation of 20 amino acids were recommended based on this work.     

Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats

It is widely recognized that activated hepatic stellate cells (HSC) play a pivotal role in development of liver fibrosis. A platelet-derived growth factor (PDGF) is the most potent mitogen for HSC. The aim of this study was to examine the effect of imatinib mesylate (STI-571, Gleevec), a clinically used PDGF receptor (PDGFR) tyrosine kinase inhibitor, on development of experimental liver fibrosis. The rat model of pig serum-induced hepatic fibrosis was used to assess the effect of daily oral administration of STI-571 on the indexes of fibrosis. STI-571 markedly attenuated development of liver fibrosis and hepatic hydroxyproline and serum fibrosis markers. The number of alpha-smooth muscle actin-positive cells and mRNA expression of alpha2-(I)-procollagen, tissue inhibitor of metalloproteinases-1, and transforming growth factor-beta were also significantly suppressed by STI-571. Our in vitro study showed that STI-571 markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner. STI-571 also significantly attenuated PDGF-BB-induced phosphorylation of PDGFR-beta, MEK1/2, and Akt in activated HSC. Because STI-571 is widely used in clinical practice, it may provide an effective new strategy for antifibrosis therapy.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.