Effects of 20-hydroxyecdysone and serotonin on neurite growth and survival rate of antennal lobe neurons in pupal stage of the silk moth Bombyx mori in vitro

Effects of 20-hydroxyecdysone and serotonin on the morphological development and the survival of antennal lobe neurons from day-2 pupal brains of the silk moth Bombyx mori were investigated in vitro. Four morphologically distinct neuronal types could be identified in the cultured antennal lobe neurons: unipolar, bipolar, multi-polar and projection neurons. Antennal lobe neurons in culture with 20-hydroxyecdysone and serotonin showed different patterns of the morphological development from those described in Manduca sexta. Projection neurons extend their neurites remarkably by 20-hydroxyecdysone in B. mori, but there is no extension from antennal lobe neurons in M. sexta. Multi-polar neurons conspicuously increase only formation of new branches from their primary neurites by serotonin in B. mori, but there are both extension and branching of the neurites in M. sexta. On day-5, antennal lobe neurons in lower titers of 20-hydroxyecdysone had significantly higher survival rates than those in higher titers. Neurons cultured for 7 days at different levels of 20-hydroxyecdysone generally showed significantly lower survival rates than neurons cultured for 5 days under the same conditions.     

Effect of resistant starch from corn or rice on glucose control,colonic events,and blood lipid concentrations in streptozotocin-induced diabetic rats

To examine the effect of two types of resistant starch on blood glucose and insulin levels, colonic events, hypolipidemic actions and humoral immune responses, Sprague-Dawley streptozotocin-induced diabetic rats were fed diet containing resistant starch from corn or rice. The marked body weight loss by inducing diabetes was not recovered by feeding resistant starch, even though there are no differences in food intakes compared to the non-diabetic control rats. No significant effect of resistant starch feeding on blood glucose and insulin was found. Even though the length of small intestines, and cecum, colon and rectum together with the tissue weight of cecum were not affected by feeding resistant starch, the intestinal transit time was markedly shortened by both types of resistant starch and resistant starch from corn had a more pronounced effect. The short chain fatty acids in the intestinal contents did not appear to be different among the groups. Nonetheless, both of resistant starch from corn and rice significantly lowered plasma total lipid and cholesterol concentrations compared to the diabetic control. The total liver cholesterol lowering effect was observed with resistant starch from rice. Neither immunoglobulin G nor C(3) were influenced by resistant starch.     

The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation

Dynamin-related GTPases regulate a wide variety of dynamic membrane processes in eukaryotes. Here, we investigated the function of ADL1C, a member of the Arabidopsis 68 kDa dynamin-like protein family. Analysis of heterozygous adl1C-1 indicates that the mutation specifically affects post-meiotic male gametogenesis. Fifty percent of the mature pollen from heterozygous adl1C-1 androecia are shriveled and fail to germinate in vitro. During microspore maturation, adl1C-1 pollen grains display defects in the plasma membrane and intine morphology, suggesting that ADL1C is essential for the formation and maintenance of the pollen cell surface and viability during desiccation. Consistent with a role in cell-surface dynamics, immunofluorescence microscopy indicates that ADL1C is localized to the cell plate of dividing somatic cells and to the tip of expanding root hairs. We propose that ADL1C functions in plasma membrane dynamics, and we discuss the role of the ADL1 family in plant growth and development.     

The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease

HslVU is a two-component ATP-dependent protease, consisting of HslV peptidase and HslU ATPase. CodW and CodX, encoded by the cod operon in Bacillus subtilis, display 52% identity in their amino acid sequences to HslV and HslU in Escherichia coli, respectively. Here we show that CodW and CodX can function together as a new type of two-component ATP-dependent protease. Remarkably, CodW uses its N-terminal serine hydroxyl group as the catalytic nucleophile, unlike HslV and certain beta-type subunits of the proteasomes, which have N-terminal threonine functioning as an active site residue. The ATP-dependent proteolytic activity of CodWX is strongly inhibited by serine protease inhibitors, unlike that of HslVU. Replacement of the N-terminal serine of CodW by alanine or even threonine completely abolishes the enzyme activity. These results indicate that CodWX in B.subtilis represents the first N-terminal serine protease among all known proteolytic enzymes.     

A novel human hydroxysteroid dehydrogenase like 1 gene (HSDL1) is highly expressed in reproductive tissuesa

We report the cloning and characterization of a novel human hydroxysteroid dehydrogenase like gene (HSDL1) located on human chromosome 16q24.2. The HSDL1 cDNA is 3407 base pair in length, encoding a 309 amino acid polypeptide related to human 17-HSD3. Northern blot reveals that the HSDL1 is highly expressed in testis and ovary. In situ hybridization indicates that the expression of HSDL1 is predominantly increased in the prostate cancer tissue compared with the normal prostate tissue, which suggests that the gene expression is important to the arising of prostate cancer.     

Monitoring the microbial contamination of beef carcass tissue with a rapid chromogenic Limulus amoebocyte lysate endpoint assay

A chromogenic Limulus amoebocyte lysate (LAL) endpoint assay was found to be an accurate and rapid means of gauging levels of beef carcass microbial contamination within 10 min. The assay demonstrated a high correlation with the total mesophilic bacterial and coliform surface populations from inoculated beef carcass surface tissues. This assay was tested on a set of actual beef carcass surface samples (n = 121) demonstrating the utility of the chromogenic LAL test as a means of monitoring carcass microbial contamination in a near real-time fashion. Classifying the chromogenic LAL results into four contamination groups was found to be a sound means of utilizing the resultant chromogenic LAL data for detecting carcasses with high levels of microbial contamination. For beef carcass testing, this assay can be used with no instrumentation other than the required 37 degrees C incubator and, as an option, a microplate reader.     

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.     

Antisense Oligonucleotide of Clusterin mRNA Induces Apoptotic Cell Death and Prevents Adhesion of Rat ASC-17D Sertoli Cells

Clusterin has been known to play important roles in cell-cell and/or cell-substratum interactions. Recently we reported the transient expression of clusterin in pancreatic endocrine cells during the early developmental stages and suggested a role in aggregating the endocrine cells for islet formation. In the present study, we have investigated the involvement of clusterin in cell-substratum interaction by the inhibition of clusterin synthesis using antisense oligonucleotide. The expression of clusterin was transiently increased as early as 2–8 h after plating the ASC-17D Sertoli cells to the culture flask, which was the period of cell attachment. In addition, up-regulation of clusterin mRNA was so much greater when the Sertoli cells were plated on the petri dish for the bacterial culture instead of in a animal cell culture flask that therefore, the cells failed to attach to it. These findings suggested that interruption of cell to plate substratum interaction might lead to over-expression of clusterin from Sertoli cells to induce cell to cell aggregation or, perhaps, to re-establish attachment with the substratum. Transfection of ASC-17D Sertoli cells with a 20-base antisense oligonucleotide against clusterin mRNA resulted in extracellular release of LDH and DNA fragmentation. Sertoli cell death by antisense oligonucleotide of clusterin was sequence specific and dose dependent. Treatment of antisense oligonucleotide induced a marked reduction of synthesis for clusterin protein, but not for clusterin mRNA expression, suggesting the translational suppression of clusterin by antisense oligonucleotide. Further, microscopic observation showed that more noticeable cell death was induced by treating the antisense prior to plating the cells than by treating after cell attachment to the plate. From these results, we speculate that down-regulation of clusterin expression in the anchorage-dependent Sertoli cells prevents them from attaching to the plate, and therefore induces cell death.     

Cytoprotective effect of arginine deiminase on taxol-induced apoptosis in DU145 human prostate cancer cells

We purified and partially sequenced a cytostatic protein from the ASC-17D Sertoli cell-conditioned media (rSCCM) showing a molecular weight of 90 kDa with homodimeric composition. N-terminal amino acid analysis revealed that the protein was homologous to the arginine deiminase (ADI) of Mycoplasma arginini. We found ADI enzyme activity in rSCCM and the abolishment of the growth inhibitory effect by the supplement of L-arginine. Thus, we confirmed that the cytostatic activity in rSCCM was due to the depletion of extracellular L-arginine by ADI. Apparent increase of cell death or DNA fragmentation was not observed in DU145 cells cultured in the presence of ADI. Incubation of DU145 cancer cells with taxol resulted in a marked DNA fragmentation, whereas pretreatment with ADI or cycloheximide protected the cells from taxol-induced apoptosis. Preincubation of the cells with ADI inhibited S35-methionine incorporation into protein synthesis in a dose dependent manner. These data suggest that ADI-induced arginine depletion may inhibit protein synthesis, and result in the protection of apoptotic cell death that requires new protein synthesis.