Molecular identification of biwa trout (Oncorhynchus masou rhodurus) using PCR-RFLP method.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to identify meat from biwa trout (Oncorhynchus masou rhodurus), amago trout (Oncorhynchus masou ishikawae), yamame trout (Oncorhynchus masou masou), and rainbow trout (Oncorhynchus mykiss). PCR amplification was conducted using primers flanking conserved regions of NADH dehydrogenase subunits 4 and 5 (ND4-ND5) (2848 bp) and ND1 (1091 bp) genes of mitochondrial DNA following restriction digestion with the enzyme HaeIII. Although the segments of ND4-ND5 and ND1 genes showed intraspecies variation, the generation of DNA fragments larger than 300 bp and 160 bp following cleavage by HaeIII of ND4-ND5 and ND1, respectively, was efficient to differentiate the four species. Furthermore, this method was successful in species identification even when using PCR-amplified products obtained from thermally processed biwa trout samples. This sensitive technique can be utilized to reveal commercial fraud, where biwa trout is adulterated with meat from cheaper counterparts.

4',6-Dimethoxyisoflavone-7-O-ß-D-glucopyranoside (wistin) is a peroxisome proliferator-activated receptor α (PPARα) agonist in mouse hepatocytes.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that regulate lipid and glucose metabolism. PPARα mainly affects fatty acid metabolism, and its activation lowers lipid levels. PPARγ is involved in the regulation of adipogenesis, insulin sensitivity, energy balance, and lipid biosynthesis. We have previously reported that 4',6-dimethoxyisoflavone-7-O-ß-D-glucopyranoside (wistin) can activate PPARγ. The purpose of the present study is to investigate the PPARα agonist activity of wistin. Using a luciferase reporter assay system of PPARα in monkey COS7 kidney cells, we showed that wistin could activate PPARα (P < 0.01 at 10 µg/mL) in a dose-dependent manner. Moreover, the addition of wistin upregulated the expression of PPARα (P < 0.01 at 10 µg/mL) and PPARα target genes including carnitine palmitoyltransferase 1a (P < 0.05 at 10 µg/mL), acyl-CoA oxidase (P < 0.01 at 10 µg/mL), acyl-CoA synthase (P < 0.05 at 10 µg/mL), PPARγ coactivator 1α (P < 0.05 at 10 µg/mL), uncoupling protein 2 (P < 0.05 at 1 µg/mL), and uncoupling protein 3 (P < 0.05 at 10 µg/mL), which are genes involved in lipid efflux and energy expenditure, in mouse primary hepatocytes. Furthermore, wistin inhibited cellular triglyceride accumulation in hepatocytes (P < 0.05 at 10 µg/mL) in a dose-dependent manner. These results indicate that wistin could suppress lipid accumulation through PPARα activation. The action of wistin on PPARα could be of interest for the amelioration of lipid metabolic disorders. To the best of our knowledge, wistin is the first reported isoflavonoid O-glycoside with PPARα agonist activity.

Sulfated Purine Alkaloid Glycosides from the Pupal Case Built by the Bruchid Beetle Bruchidius dorsalis Inside the Seed of Gleditsia japonica.

Three new sulfated isoguanine alkaloid glycosides, designated as saikachinoside A monosulfate (1), saikachinoside A disulfate (2), and locustoside B disulfate (3), have been isolated from the pupal case of the wild bruchid seed beetle Bruchidius dorsalis (Chrysomelidae, Bruchinae) infesting the seed of Gleditsia japonica Miq. (Fabaceae). Their structures were determined by spectroscopic methods and the inhibitory activity of 2 and 3 against acid phosphatase was evaluated.

Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation.

We previously revealed that endogenous bone morphogenetic protein (Bmp) activity is required for lipid accumulation in 3T3-L1 adipocytes. The present study characterized the role of endogenous Bmp activity in preadipocytes. Endogenous Bmp activity was monitored by analyzing the level of phosphorylation of Smad1/5/8, downstream molecules in the Bmp pathway. Higher levels of phosphorylated Smad1/5/8 were detected in adipogenic cells but not in non-adipogenic cells prior to differentiation induction. The inhibition of the Bmp pathway during this period decreased the expression of Pparγ2 and C/ebpα, which are transcription factors responsible for adipocyte differentiation. The expression of these transcription factors were also down-regulated by Bmp4 knockdown. In addition, endogenous Bmp4 was required for the repression of Intrleukin-11 expression. Endogenous Bmp4 in preadipocytes is indispensable for the onset of the adipogenic program, and may help to maintain the preadipocytic state during adipocyte differentiation.

Receptor expression modulates the specificity of transforming growth factor-beta signaling pathways.

In current models of transforming growth factor-beta (TGF-beta) family signaling, type II receptors activate specific activin receptor-like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand-dependent receptor regulated (R)-Smad by phosphorylating carboxy-terminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R-Smad subclasses, activin/TGF-beta-specific (AR-Smad) and bone morphogenetic protein (BMP)-specific (BR-Smad). Co-expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R-Smad subclasses in a ligand-independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF-beta1 and BMP-2 induced phosphorylation of both R-Smad subclasses, and TGF-beta1 up-regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP-2 up-regulated plasminogen activator inhibitor-1 (PAI-1), which is an AR-Smad-regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF-beta1 and BMP-2 induced phosphorylation of only the expected R-Smad. These results help to explain the diverse effects of this ligand family.

Nuclear magnetic resonance spectroscopy and mass spectrometry data for sulfated isoguanine glycosides.

The data presented here are related to the research paper entitled "Rare sulfated purine alkaloid glycosides from Bruchidius dorsalis pupal case" [1]. In this data article, we provide 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESIMS) data of three undescribed sulfated purine alkaloids, locustoside A disulfate, saikachinoside B disulfate, and saikachinoside A trisulfate isolated from the pupal case of the wild bruchid seed beetle Bruchidius dorsalis (Chrysomelidae, Bruchinae) infesting the seed of Gleditsia japonica Miquel (Fabaceae).

Establishment of a quantitative in vivo method for estimating adipose tissue volumes and the effects of dietary soy sauce oil on adipogenesis in medaka, Oryzias latipes.

Adipose tissue, which is conserved in higher eukaryotes, plays central roles in controlling the body's energy balance, including excess energy storage and energy expenditure during starvation. In adipogenesis, intranuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is a key molecule, and PPARγ agonists can promote adipogenesis. Many studies on the in vitro screening of PPARγ agonists with compounds derived from various materials have been reported; however, in vivo assays for quick examination of these feeding effects have not been established. In this study, we developed a technique using a lipophilic fluorescent reagent, Nile red to quantitatively estimate the adipose tissue volumes by using Japanese rice fish, medaka (Oryzias latipes) and studied effects of dietary soy sauce oil (SSO), which is a discarded by-product from Japanese traditional food and is known to have PPARγ-agonistic activity, on adipogenesis. We found that SSO feeding increased the adipose tissue volumes, and the expression levels of adipogenesis-related genes increased in these medaka larvae. These results suggest that SSO feeding increases the adipose tissue volumes through adipogenesis promotion by PPARγ-agonistic activity in medaka, and medaka is a powerful model for studying adipogenesis. Furthermore, our study also demonstrates the availability of SSO as a dietary additive for farmed fish.

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs.

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.

Follistatin rescues the inhibitory effect of activin A on the differentiation of bovine preadipocyte.

We investigated the effect of activin A and follistatin on the differentiation of bovine preadipocytes. Stromal-vascular (SV) cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with activin A during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. Activin A suppressed the induction of all differentiation markers regardless of the duration of treatment. The treatment with activin A also reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha mRNAs without affecting the expression of C/EBPbeta mRNA. We also observed that follistatin completely rescued the inhibitory effect of activin A on bovine preadipocyte differentiation. Furthermore, the higher doses of follistatin increased GPDH activity even in the presence of activin A compared with the cells treated with neither activin A nor follistatin. Additionally, the SV cells expressed activin A and myostatin mRNAs. These results suggest that activin A inhibits bovine preadiopocyte differentiation via affecting transcriptional cascade upstream of PPARgamma and C/EBPalpha expressions, and that follistatin suppresses the inhibitory effect of activin A on bovine preadipocyte differentiation. Endogenous activin A and/or myostatin possibly inhibit the differentiation of bovine preadipocytes.

Myostatin inhibits differentiation of bovine preadipocyte.

We investigated the effect of myostatin on the differentiation of bovine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with myostatin during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. The treatment with myostatin throughout the differentiation period severely suppressed the induction of all differentiation markers. The treatment with myostatin in the early phase of differentiation also suppressed the induction of terminal differentiation markers but three-fold higher dose of myostatin was required for the suppression compared with its treatment throughout the differentiation period. Myostatin treatment reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma mRNA and interfered with the induction of CCAAT/enhancer binding protein (C/EBP) alpha mRNA. We also observed that follistatin stimulates preadipocyte differentiation in the presence of myostatin. These results suggest that myostatin inhibits bovine preadiopocyte differentiation through suppressing PPARgamma and C/EBPalpha mRNA expressions and that follistatin counteracts the suppressive effect of myostatin.

Prenylated purine alkaloids from seeds of Gleditsia japonica.

Three previously undescribed isoguanine glycosides with an N3-prenyl group, designated locustoside B, saikachinoside B, and saikachinoside C, have been isolated from the seed of Gleditsia japonica Miquel (Fabaceae) along with two known compounds, locustoside A and saikachinoside A. Their structures were determined from spectroscopic data and X-ray crystallographic analysis. The inhibitory activity against acid phosphatase was evaluated.

4',6-dimethoxyisoflavone-7-O-ß-D-glucopyranoside (wistin) is a peroxisome proliferator-activated receptor γ (PPARγ) agonist that stimulates adipocyte differentiation.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that directly activates the expression of adipocyte-specific genes, and is universally accepted as the master regulator for adipocyte differentiation. Using a PPARγ luciferase reporter assay system, we showed that 4',6-dimethoxyisoflavone-7-O-ß-D-glucopyranoside (wistin) dose-dependently activates PPARγ. Treatment with wistin enhanced the marker of adipocyte differentiation, such as triglyceride accumulation in 3T3-L1 cells. Real-time quantitative polymerase chain reaction showed that wistin increased the expression of PPARγ2 messenger RNA. Moreover, the addition of wistin upregulated the expression of PPARγ-target genes, aP2 and adiponectin in 3T3-L1 cells. To our knowledge, wistin is the first isoflavonoid O-glycoside that exhibits PPARγ agonist activity.

Activin A inhibits differentiation of 3T3-L1 preadipocyte.

We investigated the effect of activin A on differentiation of 3T3-L1 preadipocyte. Activin A suppressed the induction of terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein (aP2) mRNA when the cells were treated with activin A throughout the differentiation period. Activin A treatment during the early phase decreased GPDH activity and aP2 mRNA level, and also reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha mRNAs without affecting the expressions of the active isoforms of C/EBPbeta and its mRNA. On the other hand, activin A treatment had no effect on the mitotic clonal expansion. These results indicate that activin A inhibits adipogenesis via affecting the transcriptional factor cascade upstream of PPARgamma expression.

PCR detection of bovine mitochondrial DNA derived from meat and bone meal in feed.

Because bovine meat and bone meal (MBM) is thought to be a major source of bovine spongiform encephalopathy, we developed a PCR-based method for detection of bovine MBM in animal feed. We isolated bone particles from feed containing bovine MBM using a separation technique based on specific gravity and then washed bone particles with sodium hypochlorite solution and an EDTA-proteinase K solution. The mitochondrial DNA was extracted from bone particles and amplified using PCR with cattle-specific primers. Bovine DNA was not detected in a milk replacer containing dried skim milk and dried whey, but bovine DNA was detected in the milk replacer that was mixed with bovine MBM. Other cattle-derived materials in feeds did not interfere with the selective detection of bovine MBM. This method allowed detection of bovine mitochondrial DNA in feed with 0.1% added bovine MBM. When the treatment with sodium hypochlorite was excluded, bovine DNA derived from MBM could not be distinguished from bovine DNA derived from other bovine materials. However, the exclusion of this treatment improved the detection limit of bovine MBM in feed. This method appears suitable for the selective detection of bovine MBM in feed.

Nitric oxide suppresses preadipocyte differentiation in 3T3-L1 culture.

Nitric oxide (NO) is an important chemical messenger controlling many physiological functions, involving cell proliferation, and differentiation. The purpose of this study was to investigate the effect of NO on adipocyte differentiation using a murine preadipocyte cell line, 3T3-L1. The treatment with a NO donor, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), reduced some markers of adipocyte differentiation such as glycerol-3-phosphate dehydrogenase activity, and intracellular lipid accumulation. To examine whether these effects of NOC18 on adipocyte differentiation markers are due to its cytotoxity, lactate dehydrogenase (LDH) release from the cells were measured. NOC18 did not affect LDH release into the culture medium. Thus, the suppressive actions of NO donor were unlikely to result from its cytotoxicity. Peroxisome proliferator-activated receptor (PPAR) gamma is a critical transcription factor for adipocyte differentiation and adipocyte fatty acid binding protein (aP2) gene is one of its targets. Protein expression of PPARgamma was not diminished by NOC18 treatment, although mRNA expression of aP2 was reduced. Electrophoretic mobility shift assay showed that NOC18 interfered with the DNA binding activity of PPARgamma. Therefore, the present experiment suggest that NO suppresses adipocyte differentiation through suppressing the transcriptional activity of PPARgamma, without suppressing its expression level.

Yeast substrate-trapping system for isolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine phosphatase receptor type z.

Although members of the protein tyrosine phosphatase (PTP) family are known to play critical roles in various cellular processes through the regulation of protein tyrosine phosphorylation in cooperation with protein tyrosine kinases (PTKs), the physiological functions of individual PTPs are poorly understood. This is due to a lack of information concerning the physiological substrates of the respective PTPs. Several years ago, substrate-trap mutants were developed to identify the substrates of PTPs, but only a limited number of PTP substrates have been identified using typical biochemical techniques in vitro. The application of this strategy to all the PTPs seems difficult, because the substrates identified to date were restricted to relatively abundant and highly tyrosine phosphorylated cellular proteins. Therefore, the development of a standard method applicable to all PTPs has long been awaited. We report here a genetic method to screen for PTP substrates which we have named the "yeast substrate-trapping system." This method is based on the yeast two-hybrid system with two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate the prey protein, and screening using a substrate-trap PTP mutant as bait. This method is probably applicable to all the PTPs, because it is based on PTP-substrate interaction in vivo, namely the substrate recognition of individual PTPs. Moreover, this method has the advantage that continuously interacting molecules for a PTP are also identified, at the same time, under PTK-noninductive conditions. The identification of physiological substrates will shed light on the physiological functions of individual PTPs.