Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.     

Age-related changes of serum lipoprotein oxidation in rats

Oxidation of low-density lipoprotein (LDL) may be a prelude to atherogenesis and directly age related. To assess whether there may be relationship between age and plasma lipoprotein (LP) oxidation, we studied copper-mediated LP oxidation isolated from the blood of 2 months, 7 months, and 15 months old rats. We determined whether the susceptibility of LP to oxidation might be related to vitamin C levels in serum, vitamin E levels in LP, or the total antioxidant capacity (TAC) of serum or LP. Serum vitamin C content was inversely related to age, malondialdehyde (MDA) propagation rate, and maximum change of MDA concentrations. However, there were no significant relationships between age and serum TAC, LP TAC, serum vitamin E, or the ratio of LP vitamin E to serum vitamin C content. The lag phase of MDA formation was significantly decreased with age and the ratio of LP vitamin E content to serum vitamin C content, increased with age. Maximum change of MDA concentration was positively correlated with the ratio of LP vitamin E contents to serum vitamin C concentration. Thus, as the rat ages, vitamin C status decreases with an increased LP susceptibility to oxidation. It is tempting to speculate that enhanced LP oxidation in older rats may reflect a reduced amount of recycling of LDL vitamin E by serum vitamin C.     

A major Li(+) extrusion system NhaB of Pseudomonas aeruginosa : comparison with the major Na(+) extrusion system NhaP

A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters. We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.     

Antitumor activity and action mechanisms of Lactobacillus casei through the regulation of immune responses

Lactobacillus casei strain Shirota (LcS) has been demonstrated to have beneficial effects in numerous disease models, especially murine autoimmune disease and carcinogenesis models, via host immune modulation. It has also been reported that LcS induced recovery of the host immune responses that were decreased by treatment with carcinogens, and that augmented the natural killer activity and T cell functions of host immune cells. After LcS is ingested by the host, it is incorporated into M cells in Peyer's patches (PP) and digested to form active components. In PP, macrophages or dendritic cells that phagocytosed LcS gained ability to produce tumor necrosis factor-alpha. The components of LcS digested in PP were then recognized through toll-like receptor 2 in macrophages, resulting in the production of several cytokines which elicit varied responses in host immune cells. Also, it was observed by 2D-PAGE analyses that the expression level and/or the phosphorylation of some proteins in PP and mesenteric lymph nodes were definitely altered after the ingestion of LcS, providing more evidence of cellular responses.     

Simple and rapid determination of 1-deoxynojirimycin in mulberry leaves

A simple and rapid method for determining 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor present in mulberry leaves (Morus alba and Morus bombysis), by high performance liquid chromatography coupled to an evaporative light scattering detector (ELSD) has been developed. DNJ was separated from extract of mulberry leaves on TSK gel Amide-80 column, which is a representative column for hydrophilic interaction chromatography. During post column detection, DNJ was detected by ELSD and concurrently identified by mass spectrometry. The detection limit was 100 ng. This method is sufficiently sensitive for determining DNJ in mulberry leaves and other related products.     

Efflux of sphingoid bases by P-glycoprotein in human intestinal Caco-2 cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 microM digoxin for 48 h caused up-regulation of multidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.     

Multidrug-resistant cancer cell susceptibility to cytotoxic quassinoids, and cancer chemopreventive effects of quassinoids and canthin alkaloids

Twenty-three quassinoids (1-23), which were isolated previously from Simaroubaceous plants, were evaluated for cytotoxicity against three multidrug-resistant cancer cell lines, KB-VIN, KB-7d, and KB-CPT. Nine compounds (2-7 and 9-11) showed significant cytotoxicity in all three cell lines. Compounds 1, 12-14, 17, and 20 demonstrated significant activity against the KB-7d and KB-CPT cell lines, and compounds 18, 19, and 23 revealed notable activity only against KB-7d cells. Structure-activity relationships were drawn based on these data. In addition, six quassinoid derivatives (24-29) and four canthin alkaloids (30-33), which were isolated from Brucea antidysenterica, were examined for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus early antigen (EBV-EA) activation as cancer chemopreventive agents. All of these compounds demonstrated significant inhibitory effects against EBV-EA activation.     

Reference gene selection for real-time RT-PCR in regenerating mouse livers

The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.     

Complete genome sequence of Melissococcus plutonius DAT561, a strain that shows an unusual growth profile and is representative of an endemic cluster in Japan

We report the complete genome sequence of Melissococcus plutonius DAT561, which is a causative agent of European foulbrood. M. plutonius DAT561 is a representative of nonfastidious strains isolated in Japan. The addition of potassium phosphate was not required for normal growth, unlike for typical M. plutonius strain/isolates.