A novel class of apical sodium-dependent bile acid transporter inhibitors: the amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinoline derivatives

A series of 4-oxo-1-phenyl-1,4-dihydroquinolines possessing a linker and an ammonio moiety were synthesized and found to inhibit the apical sodium-dependent bile acid transporter (ASBT). The potency of ASBT inhibition varied with the position and length of the linking tether. Compound 21e effectively lowered the total serum cholesterol levels in hamsters.     

Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation

We previously targeted EGFP (a mutant of green fluorescent protein) to the lumen of the mouse sperm acrosome and reported the time course of EGFP release during the acrosome reaction. In the study reported here, we estimated the pH within the mouse sperm acrosome utilizing the pH-dependent nature of EGFP fluorescence. The average intra-acrosomal pH was estimated to be 5.3 +/- 0.1 immediately after sperm preparation, gradually increasing to 6.2 +/- 0.3 during 120 min of incubation in TYH media suitable for capacitation. Spontaneous acrosome reactions were noted to increase concomitantly with acrosomal alkalinization during incubation. We also demonstrated that acrosomal antigens detected by monoclonal antibodies MN7 and MC41 did not dissolve following the acrosome reaction in pH 5.3 media, but dissolved at pH 6.2. These data suggest that acrosomal alkalinization during incubation conducive for sperm capacitation may function to alter acrosomal contents and prepare them for release during the acrosome reaction.     

Complementary DNA cloning and characterization of rat spergen-2, a spermatogenic cell-specific gene 2 encoding a 56-kilodalton nuclear protein bearing ankyrin repeat motifs

Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1-6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX-XIV) and spermatids (steps 1-11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.     

2'-benzyloxychalcone derivatives stimulate glucose uptake in 3T3-L1 adipocytes

By a cell-based glucose uptake screening assay, a chalcone derivative, 3-nitro-2'-benzyloxychalcone (compound 1) was identified. Compound 1 stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. When cells were treated with various concentrations of insulin in the presence of compound 1, marked enhancement of insulin-stimulated glucose uptake was observed at each concentration, suggesting that the compound might function as an insulin sensitizer. Preliminary study on the structure-activity relationships revealed that two aromatic benzene rings tolerated several substituents, but substitution by acidic or highly polar groups abolished the activity. Among several chalcone derivatives, 4-chloro-2'-benzyloxychalcone (compound 8) showed the highest level of activity. Compound 8-stimulated glucose uptake was almost completely inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). These results suggest that the action of chalcone derivatives is mediated via a pathway involving PI3K.     

Mouse germ cell-less as an essential component for nuclear integrity

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2 beta (lamina-associated polypeptide 2 beta). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.     

Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium

An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.     

Preferential localization of SSEA-4 in interfaces between blastomeres of mouse preimplantaion embryos

The monoclonal antibody 6E2 raised against the embryonal carcinoma cell line NCR-G3 had been shown to also react with human germ cells. Thin-layer chromatography (TLC) immunostaining revealed that 6E2 specifically reacts with sialosylglobopentaosylceramide (sialylGb5), which carries an epitope of stage-specific embryonic antigen-4 (SSEA-4), known as an important cell surface marker of embryogenesis. The immunostaining of mouse preimplantation embryos without fixation showed that the binding of 6E2 caused the clustering and consequent accumulation of sialylGb5 at the interface between blastomeres. These results suggest that SSEA-4 actively moves on the cell surface and readily accumulates between blastomeres after binding of 6E2.     

Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.     

An inducer of VGF protects cells against ER stress-induced cell death and prolongs survival in the mutant SOD1 animal models of familial ALS

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease, and recent evidence has suggested that endoplasmic reticulum (ER) stress signaling is involved in the pathogenesis of ALS. Here we identified a small molecule, SUN N8075, which has a marked protective effect on ER stress-induced cell death, in an in vitro cell-based screening, and its protective mechanism was mediated by an induction of VGF nerve growth factor inducible (VGF): VGF knockdown with siRNA completely abolished the protective effect of SUN N8075 against ER-induced cell death, and overexpression of VGF inhibited ER-stress-induced cell death. VGF level was lower in the spinal cords of sporadic ALS patients than in the control patients. Furthermore, SUN N8075 slowed disease progression and prolonged survival in mutant SOD1 transgenic mouse and rat models of ALS, preventing the decrease of VGF expression in the spinal cords of ALS mice. These data suggest that VGF plays a critical role in motor neuron survival and may be a potential new therapeutic target for ALS, and SUN N8075 may become a potential therapeutic candidate for treatment of ALS.