Identification of (Na,K)ATPase inhibitor in brine shrimp,Artemia salina,as long-chain fatty acids

Summary An inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.Abbreviations ATPase adenosine triphosphatase - EDTA ethylenediamine-tetraacetate - TLC thin-layer chromatography     

A novel MHC class I-related molecule expressed on mouse thymic stroma cells and mature lymphocytes

A monoclonal antibody (mAb) TP-3 has been established by immunizing rats with the BALB/c mouse thymic epithelial cell line TEL-2. The TP-3 antigen is expressed on stroma cells of thymus, spleen, and lymph node in syngeneic BALB/c mice (H-2 ^d ). This antigen is also expressed at a low level on the cell surface of immature thymocytes, and at a high level on mature T and B cells. In allogeneic mice such as C57BL/6 (H-2 ^b ) or C3H (H-2 ^k ), no cells expressed the TP-3 antigen. Using H-2 congenic mice, reactivity with mAb TP-3 was found to map to a region of H-2D ^d L ^d or between D ^d and Qa, suggesting that TP-3 is a major histocompatibility complex (MHC) class I antigen. However, immunoprecipitation analysis indicated that this antigen is not identical to the classical mouse class I molecules in terms of molecular size, antigenicity, and tissue distribution.     

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; i.e., ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.     

RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major pathways that account for survival after post-replicational DNA damage. TLS functions by filling gaps on a daughter strand that remain after DNA replication caused by damage on the mother strand, while HR can repair gaps and breaks using the intact sister chromatid as a template. The RAD18 gene, which is conserved from lower eukaryotes to vertebrates, is essential for TLS in Saccharomyces cerevisiae. To investigate the role of RAD18, we disrupted RAD18 by gene targeting in the chicken B-lymphocyte line DT40. RAD18(-/-) cells are sensitive to various DNA-damaging agents including ultraviolet light and the cross-linking agent cisplatin, consistent with its role in TLS. Interestingly, elevated sister chromatid exchange, which reflects HR- mediated post-replicational repair, was observed in RAD18(-/-) cells during the cell cycle. Strikingly, double mutants of RAD18 and RAD54, a gene involved in HR, are synthetic lethal, although the single mutant in either gene can proliferate with nearly normal kinetics. These data suggest that RAD18 plays an essential role in maintaining chromosomal DNA in cooperation with the RAD54-dependent DNA repair pathway.     

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled,porcine male-specific DNA probe produced by polymerase chain reaction

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5′-AAGTGGTCAGCGTGTCCATA-3′ and 5′-TTTCTCCTGTATCCTCCTGC-3′) for 236 bp fragment of porcine male-specific DNA sequence and 1.25 × 10⁴ template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization. © 1995 Wiley-Liss, Inc.     

Two new phenolic glucosides and an ellagitannin from the leaves of Castanopsis sclerophylla

In the course of a chemotaxonomical study of Castanopsis species (Fagaceae), detailed investigation of the leaves of C. sclerophylla led to isolation of three new phenolic compounds together with 62 known compounds. The structures of the new compounds were elucidated as 2-O-galloyl-O-4,6-(S)-valoneoyl-d-glucose (1), 6-O-galloyl-1-O-vanilloyl-β-d-glucose (2), and 4″-O-galloylchestanin (3) by means of spectroscopic analyses and enzymatic hydrolysis with tannase. Comparison with other Castanopsis species indicated that C. sclerophylla characteristically accumulates chlorogenic acid and a dimeric ellagitannin, rugosin E. Triterpene hexahydroxydiphenoyl esters, which are major constituents of C. cuspidata var. sieboldii, C. hystrix, and C. fissa were not detected.     

Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.