TAT1 and TAT2 tyrosine aminotransferases have both distinct and shared functions in tyrosine metabolism and degradation in Arabidopsis thaliana.

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower 13C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

DNA marker-assisted evaluation of cooked bean hardness of three soybean progeny lines.

Cooked bean hardness is an important trait for the processing of soybean products such as nimame, natto, miso, and soy sauce. Previously, we showed that cooked bean hardness is primarily affected by the pectin methylesterase gene Glyma03g03360, and that calcium content has a secondary effect on this trait. To establish a simple and timely method for the evaluation of cooked bean hardness, primers of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were designed to detect a single-nucleotide polymorphism of Glyma03g03360 and subsequently used to evaluate three soybean progeny lines. The determined genotypes were compared to those identified using the cleaved amplified polymorphic sequence (CAPS) method. Seven out of 284 lines presented different genotypes, which were determined using the two methods: A genotypes were incorrectly assigned as heterozygous by CAPS, suggesting that ARMS-PCR is more reliable. Glyma03g03360 genotypes could be used to evaluate cooked bean hardness, except for intermediate values. Cooked bean hardness within the same genotype groups was significantly correlated with calcium contents. These findings indicate that ARMS-PCR is useful for a marker-assisted selection of soybean with soft-cooked beans and that calcium content may be used for additional selection.

Expression-based genome-wide association study links the receptor CD44 in adipose tissue with type 2 diabetes.

Type 2 diabetes (T2D) is a complex, polygenic disease affecting nearly 300 million people worldwide. T2D is primarily characterized by insulin resistance, and growing evidence has indicated the causative link between adipose tissue inflammation and the development of insulin resistance. Genetic association studies have successfully revealed a number of important genes consistently associated with T2D to date. However, these robust T2D-associated genes do not fully elucidate the mechanisms underlying the development and progression of the disease. Here, we report an alternative approach, gene expression-based genome-wide association study (eGWAS): searching for genes repeatedly implicated in functional microarray experiments (often publicly available). We performed an eGWAS across 130 independent experiments (totally 1,175 T2D case-control microarrays) to find additional genes implicated in the molecular pathogenesis of T2D and identified the immune-cell receptor CD44 as our top candidate (P = 8.5 × 10(-20)). We found CD44 deficiency in a diabetic mouse model ameliorates insulin resistance and adipose tissue inflammation and also found that anti-CD44 antibody treatment decreases blood glucose levels and adipose tissue macrophage accumulation in a high-fat, diet-fed mouse model. Further, in humans, we observed CD44 is expressed in inflammatory cells in obese adipose tissue and discovered serum CD44 levels were positively correlated with insulin resistance and glycemic control. CD44 likely plays a causative role in the development of adipose tissue inflammation and insulin resistance in rodents and humans. Genes repeatedly implicated in publicly available experimental data may have unique functionally important roles in T2D and other complex diseases.

Linkage mapping, molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase.

There are substantial genotypic differences in the levels of flavonol glycosides (FGs) in soybean leaves. The first objective of this study was to identify and locate genes responsible for FG biosynthesis in the soybean genome. The second objective was to clone and verify the function of these candidate genes. Recombinant inbred lines (RILs) were developed by crossing the Kitakomachi and Koganejiro cultivars. The FGs were separated by high performance liquid chromatography (HPLC) and identified. The FGs of Koganejiro had rhamnose at the 6″-position of the glucose or galactose bound to the 3-position of kaempferol, whereas FGs of Kitakomachi were devoid of rhamnose. Among the 94 RILs, 53 RILs had HPLC peaks classified as Koganejiro type, and 41 RILs had peaks classified as Kitakomachi type. The segregation fitted a 1:1 ratio, suggesting that a single gene controls FG composition. SSR analysis, linkage mapping and genome database survey revealed a candidate gene in the molecular linkage group O (chromosome 10). The coding region of the gene from Koganejiro, designated as GmF3G6″Rt-a, is 1,392 bp long and encodes 464 amino acids, whereas the gene of Kitakomachi, GmF3G6″Rt-b, has a two-base deletion resulting in a truncated polypeptide consisting of 314 amino acids. The recombinant GmF3G6″Rt-a protein converted kaempferol 3-O-glucoside to kaempferol 3-O-rutinoside and utilized 3-O-glucosylated/galactosylated flavonols and UDP-rhamnose as substrates. GmF3G6″Rt-b protein had no activity. These results indicate that GmF3G6″Rt encodes a flavonol 3-O-glucoside (1 â†’ 6) rhamnosyltransferase and it probably corresponds to the Fg2 gene. GmF3G6″Rt was designated as UGT79A6 by the UGT Nomenclature Committee.

Novel Soybean Variety Lacking Raffinose Synthase 2 Activity.

Variation in the raffinose family oligosaccharide (RFO) content in soybean is advantageous for livestock farming and health science. In this study, a soybean variety (GmJMC172) with a significantly low stachyose content in its seeds was identified in the NARO Genebank core collection. The results of the single-nucleotide polymorphism (SNP) analysis suggested that this phenomenon was related to a single-base deletion, inducing a frameshift mutation in raffinose synthase 2 (RS2), rather than the polymorphisms in the RS3, RS4, and stachyose synthase (STS) sequences. Differences in the enzymatic properties between the native RS2 and truncated RS2 were examined by using a three-dimensional model predicted using Alphafold2. In addition to revealing the missing active pocket in truncated RS2, the modeled structure explained the catalytic role of W331* and suggested a sufficient space to bind both sucrose and raffinose in the ligand-binding pocket. The soybean line, with seeds available from the NARO Genebank, could serve as breeding materials for manipulating the RFO content.

The soybean F3'H protein is localized to the tonoplast in the seed coat hilum.

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Combination of anti-CD25 antibody and poly I:C treatment in pregnant NOD mice may be used as 'pregnancy-related' type 1 diabetes model.

AIMS/INTRODUCTION: Some women develop type 1 diabetes during pregnancy or immediately after delivery. However, the underlying pathophysiology remains largely unknown, probably because of the lack of a suitable animal model. In this study, we administered pregnant NOD mice with an anti-CD25 antibody to reduce regulatory T cells along with poly I:C and examined the onset of diabetes. MATERIALS AND METHODS: Anti-CD25 antibody and poly I:C were intraperitoneally administered to mated female NOD mice. Mice delivered within 3 weeks after the treatment, and the onset of diabetes during pregnancy or within 6 weeks after delivery was examined. Some mice were killed 1 week after treatment, and their spleen and pancreas were excised to examine the expression levels of cytokines and for histological examination. RESULTS: Half of the mice treated with anti-CD25 antibody plus poly I:C developed diabetes, as compared with none of the poly I:C-injected mice (P < 0.05). The ratios of interleukin-18/forkhead box P3 and granzyme B/forkhead box P3 were higher in the pancreas of anti-CD25 antibody plus poly I:C-treated mice than in the pancreas of control mice. The insulitis score decreased in the pancreas of anti-CD25 antibody plus poly I:C-injected pregnant NOD mice. CONCLUSIONS: We describe the use of anti-CD25 antibody to reduce regulatory T cells and poly I:C as a Toll-like receptor 3 stimulator to mimic viral infection in a pregnant NOD mouse, which can be used as a model of 'pregnancy-related' type 1 diabetes.

Expression-based Genome-wide Association Study Links OPN and IL1-RA With Newly Diagnosed Type 1 Diabetes in Children.

CONTEXT: Islet autoantibodies (IAbs) are currently the most reliable indicators of islet autoimmunity. However, IAbs do not fully meet the need for the prediction and intervention of type 1 diabetes (T1D). Serological proteins should be great sources for biomarkers. OBJECTIVE: This work aimed to identify new proteomic biomarkers with the technology of an expression-based genome-wide association study (eGWAS) in children newly diagnosed with T1D. METHODS: In an attempt to identify additional biomarkers, we performed an eGWAS using microarray data from 169 arrays of the pancreatic islets of T1D rodents (78 T1D cases and 91 controls). We ranked all 16 099 protein-coding genes by the likelihood of differential expression in the pancreatic islets. Our top 20 secreted proteins were screened in 170 children including 100 newly diagnosed T1D, and 50 type 2 diabetes (T2D) and 20 age-matched healthy children. With 6 proteins showing significance, we further conducted a validation study using the second independent set of 400 samples from children including 200 newly diagnosed with T1D, 100 T2D, and 100 age-matched controls. RESULTS: We identified 2 serum proteins that were significantly changed in T1D vs both control and T2D, and 5 serum proteins were significantly changed both in T1D and T2D vs control. Serum osteopontin (OPN) levels were uniquely higher in T1D (T1D vs controls, P = 1.29E-13 ~ 9.38E-7, T1D vs T2D, P = 2.65E-8 ~ 1.58E-7) with no difference between T2D and healthy control individuals. Serum interleukin 1 receptor antagonist (IL-1RA) levels were lower in T1D compared both with T2D (P = 3.36E-9~0.0236) and healthy participants (P = 1.09E-79 ~ 2.00E-12). CONCLUSION: Our results suggest that OPN and IL1-RA could be candidates for useful biomarkers for T1D in children.

Difference in chilling-induced flavonoid profiles, antioxidant activity and chilling tolerance between soybean near-isogenic lines for the pubescence color gene.

Chilling tolerance is an important trait of soybeans [Glycine max (L.) Merr.] produced in cool climates. We previously isolated a soybean flavonoid 3' hydroxylase (F3'H) gene corresponding to the T locus, which controls pubescence and seed coat color. A genetic link between the T gene and chilling tolerance has been reported, although the exact underlying mechanisms remain unclear. Using the soybean near-isogenic lines (NILs) To7B (TT) and To7G (tt), we examined the relationship between chilling injury, antioxidant activity and flavonoid profiles associated with chilling treatment (15°C). Chilling injury was more severe in the second trifoliate leaves of To7G than in those of To7B. Hydrogen peroxide accumulation and lipid peroxidation were enhanced by chilling in To7G. Chilling-induced enhancement of antioxidant activity was more prominent in To7B than in To7G. High performance liquid chromatography analysis indicated that the contents of quercetin glycosides and isorhamnetin glycosides (3',4'-dihydroxylated flavonol derivatives) increase in the second trifoliate leaves of To7B after chilling treatment, whereas the same treatment increased kaempferol glycoside (4'-monohydroxylated flavonol derivatives) content in the corresponding leaves of To7G. Histochemical staining also demonstrated chilling-induced flavonoid accumulation. Microarray analysis and real-time reverse transcription-PCR demonstrated that the transcript levels of soybean F3'H are upregulated by chilling. The differences in chilling injury, antioxidant activity and flavonoid species between the two NILs support the notion that soybean F3'H affects chilling tolerance by increasing antioxidant activity via production of 3',4'-dihydroxylated flavonol derivatives.

Pentoxifylline prevents pig serum-induced rat liver fibrosis by inhibiting interleukin-6 production.

BACKGROUND/AIM: Pig serum-induced rat liver fibrosis is a model of liver fibrosis in the absence of obvious hepatocyte injury. Penoxifylline (PTX), a xanthine derivative, which is a well-known suppressor of tumor necrosis factor-alpha (TNF-alpha) production from inflammatory cells, has also been shown to inhibit the growth of hepatic stellate cells and to inhibit collagen synthesis in these cells in vitro. We investigated the effect of PTX on pig serum-induced liver fibrosis in vivo, and assessed the mechanisms of prevention of fibrogenesis by this drug. METHODS: Male Wistar rats were given intraperitoneal injections of 0.5 ml normal pig serum twice a week for 10 weeks with or without concomitant oral administration of PTX (20 mg/kg). RESULTS: Rats that received pig serum showed significant liver fibrosis, and their serum interleukin-6 (IL-6) and hyaluronic acid levels were significantly increased. The serum levels of IL-6 were well correlated with the serum levels of hyaluronic acid, and increased as the liver fibrosis progressed. Penoxifylline prevented the development of fibrosis in this animal model and reduced the serum levels of IL-6 in a dose-dependent manner. In vitro, by the addition of PTX to the culture medium of the rat hepatic stellate cells (HSCs), the proliferation of the HSCs was significantly inhibited and IL-6 in the culture supernatant was also reduced significantly. Exogenous addition of IL-6 partially restored the proliferation. CONCLUSION: Penoxifylline prevents pig serum-induced rat liver fibrosis by inhibiting the proliferation of HSCs and by inhibiting the production of IL-6 from HSCs.

Telmisartan but not candesartan affects adiponectin expression in vivo and in vitro.

To examine the effects of telmisartan on peroxisome proliferator-activated receptor gamma activation, we compared the effects of telmisartan with those of candesartan on adipocytokines and glucose and lipid metabolism in vivo and in vitro. In vivo, 56 patients with both type 2 diabetes and hypertension were enrolled and randomized to receive either telmisartan (40 mg) or candesartan (8 mg) for 3 months. Serum adiponectin, HbA1c levels, lipid profiles and blood pressure were recorded at the beginning and 3 months later. In vitro, differentiated 3T3-L1 adipocytes were treated with telmisartan, candesartan, pioglitazone or vehicle for 24 h, and then adiponectin mRNA and protein levels were measured. The results showed that most of the metabolic parameters, including the lipid profiles, did not change significantly during the study in either group. However, the changes in serum adiponectin and plasma glucose over 3 months were significantly greater in the telmisartan group than in the candesartan group. In vitro, although the protein level of adiponectin was not significantly elevated, the mRNA expression of adiponectin was elevated 1.5-fold by telmisartan in 3T3-L1 adipocytes. Our findings suggest that telmisartan may have beneficial effects in type 2 diabetes beyond its antihypertensive effect.

Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND AND AIM: The effect of polaprezinc, a zinc-carnosine chelate compound, on the development of non-alcoholic steatohepatitis (NASH) was investigated in dietary methionine and choline deficient (MCD) mice. METHODS: Mice were fed the MCD diet with or without polaprezinc (2.2 g/kg diet) for 10 weeks. Liver histopathology, triglyceride and lipid peroxide levels, and the expression of genes linked to fibrosis were then assessed. RESULTS: MCD mice developed steatohepatitis accompanied by mild fibrosis with an increase in lipid peroxidation, hepatic stellate cell (HSC) activation, and the augmented mRNA expression of tumor necrosis factor-alpha, transforming growth factor-beta1 and procollagen alpha1(I). The mRNA expression levels of matrix metalloproteinase (MMP)-2 and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also enhanced. Histopathologically, polaprezinc supplementation did not influence the development of steatosis but it apparently attenuated fibrosis. Polaprezinc slightly reduced lipid peroxidation and suppressed HSC activation as well as the mRNA expression of pro-inflammatory cytokines. Polaprezinc affected the MCD diet-enhanced expression of TIMP-1 even when administered relatively late. CONCLUSION: These results suggest that polaprezinc attenuates fibrosis in NASH by reducing inflammation and lipid peroxidation and, during a later phase, promoting fibrolysis via the inhibition of TIMP expression in the liver. Further investigation is required to clarify the clinical efficacy of polaprezinc in patients with NASH.

Relationship between protein composition and coagulation reactivity, particulate formation, and incorporation of lipids in soymilk.

Many studies have suggested that the 11S/7S ratio in soybeans affects the coagulation reaction at the first step. In this study, the 11S/7S ratio in soybeans showed significantly negative correlation with MgCl(2) concentrations for the maximum breaking stress of tofu for six Japanese varieties. To determine the effect of the 11S/7S ratio, soymilk was fractionated by centrifugation after the addition of MgCl(2), and the distribution of lipids and proteins was studied. The amount of precipitate increased as the MgCl(2) concentration or the 11S/7S ratio increased. More triglyceride was incorporated into the precipitate as the MgCl(2) concentration or the 11S/7S ratio increased. The stain intensity of bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the ratio of oleosin, a membrane protein of the oil body, increased in the precipitate as the MgCl(2) concentration or the 11S/7S ratio increased, while the ratios of glycinin and beta-conglycinin were less variable. These results indicate that the 11S/7S ratio and coagulant concentration may have an effect on the amount of coagulum and the concentration of oil globules in the coagulum at the beginning of coagulation.

Cyclooxygenase-2 inhibitor and interferon-beta synergistically induce apoptosis in human hepatoma cells in vitro and in vivo.

Recent clinical trials have shown that interferon (IFN) is effective for chemoprevention against hepatocellular carcinoma (HCC). However, it remains controversial as to whether IFN exerts direct cytotoxicity against HCC. Cyclooxygenase (COX)-2 also plays a role in hepatocarcinogenesis and may mediate resistance to apoptosis in HCC. Therefore, we aimed to elucidate the combined effect of COX-2 inhibitor, NS-398, and IFN on in vitro growth suppression of HCC using 3 hepatoma cell lines (HepG2, PLC/PRF/5, and Huh7) and in vivo nude mouse xenotransplantation model using Huh7 cells. Only minimal growth inhibition was observed after treatment with IFN-beta alone in the 3 hepatoma cell lines. In contrast, treatment with NS-398 and IFN-beta synergistically inhibited cell proliferation in dose- and time-dependent manner. Apoptosis was identified by 4',6-diamidino-2-phenylindole dihydrochloride and fluorescent staining. IFN-beta up-regulated the expression of TRAIL, while NS-398 increased the expression of TRAIL receptors (especially of death receptor 5). Subsequently, activation of caspase-8 and caspase-3 was observed following the treatment with NS-398 and IFN-beta. Blockade of TRAIL with a specific antibody attenuated this apoptosis. Furthermore, we found that IFN-beta up-regulated COX-2 expression in Huh7 cells, and NS-398 might suppress the up-regulated COX-2 activity downstream of IFN signaling. In vivo experiment showed the combined regimen with NS-398 and IFN-beta reduced the growth of xenotransplated HCCs in nude mice. In conclusion, NS-398 is sufficient to overcome IFN resistance in hepatoma cells through the TRAIL/TRAIL receptor pathway, therefore, the combination would appear to be a new therapeutic regimen for HCC.

Biochemical properties and subcellular localization of tyrosine aminotransferases in Arabidopsis thaliana.

Plants produce various L-tyrosine (Tyr)-derived compounds that are of pharmaceutical or nutritional importance to humans. Tyr aminotransferase (TAT) catalyzes the reversible transamination between Tyr and 4-hydroxyphenylpyruvate (HPP), the initial step in the biosynthesis of many Tyr-derived plant natural products. Herein reported is the biochemical characterization and subcellular localization of TAT enzymes from the model plant Arabidopsis thaliana. Phylogenetic analysis showed that Arabidopsis has at least two homologous TAT genes, At5g53970 (AtTAT1) and At5g36160 (AtTAT2). Their recombinant enzymes showed distinct biochemical properties: AtTAT1 had the highest activity towards Tyr, while AtTAT2 exhibited a broad substrate specificity for both amino and keto acid substrates. Also, AtTAT1 favored the direction of Tyr deamination to HPP, whereas AtTAT2 preferred transamination of HPP to Tyr. Subcellular localization analysis using GFP-fusion proteins and confocal microscopy showed that AtTAT1, AtTAT2, and HPP dioxygenase (HPPD), which catalyzes the subsequent step of TAT, are localized in the cytosol, unlike plastid-localized Tyr and tocopherol biosynthetic enzymes. Furthermore, subcellular fractionation indicated that, while HPPD activity is restricted to the cytosol, TAT activity is detected in both cytosolic and plastidic fractions of Arabidopsis leaf tissue, suggesting that an unknown aminotransferase(s) having TAT activity is also present in the plastids. Biochemical and cellular analyses of Arabidopsis TATs provide a fundamental basis for future in vivo studies and metabolic engineering for enhanced production of Tyr-derived phytochemicals in plants.

Expression-Based Genome-Wide Association Study Links Vitamin D-Binding Protein With Autoantigenicity in Type 1 Diabetes.

Type 1 diabetes (T1D) is caused by autoreactive T cells that recognize pancreatic islet antigens and destroy insulin-producing ß-cells. This attack results from a breakdown in tolerance for self-antigens, which is controlled by ectopic antigen expression in the thymus and pancreatic lymph nodes (PLNs). The autoantigens known to be involved include a set of islet proteins, such as insulin, GAD65, IA-2, and ZnT8. In an attempt to identify additional antigenic proteins, we performed an expression-based genome-wide association study using microarray data from 118 arrays of the thymus and PLNs of T1D mice. We ranked all 16,089 protein-coding genes by the likelihood of finding repeated differential expression and the degree of tissue specificity for pancreatic islets. The top autoantigen candidate was vitamin D-binding protein (VDBP). T-cell proliferation assays showed stronger T-cell reactivity to VDBP compared with control stimulations. Higher levels and frequencies of serum anti-VDBP autoantibodies (VDBP-Abs) were identified in patients with T1D (n = 331) than in healthy control subjects (n = 77). Serum vitamin D levels were negatively correlated with VDBP-Ab levels in patients in whom T1D developed during the winter. Immunohistochemical localization revealed that VDBP was specifically expressed in α-cells of pancreatic islets. We propose that VDBP could be an autoantigen in T1D.

New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet.

We have developed a comprehensive information platform, named KeyMolnet, for drug discovery and life science research in the post-genome era. Using KeyMolnet, we show new approaches to research into the biological mechanism in DNA microarray analysis. Thanks to the DNA microarray technology, it is now possible to obtain very large quantities of gene expression data at a time. However, it is still difficult to extract meaningful information from such large quantities of data and to analyze the relationship between gene expression data and biological function. We therefore developed an advanced tool that can generate molecular networks upon demand, and beyond signaling "cross-talks," can connect them to physiological phenomena and medical and drug information. Here we show the methods of mechanism analysis using the DNA microarray data and KeyMolnet, as well as the possible mechanism of inducing apoptosis in the human promyelocytic leukemia cell line, HL-60, treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), using the time series of gene expression data from DNA microarray experiments. KeyMolnet enables practical approaches to research into biological mechanisms, which in turn contribute to new discoveries in the medical, pharmaceutical and life sciences.

Anti-CD44 antibody treatment lowers hyperglycemia and improves insulin resistance, adipose inflammation, and hepatic steatosis in diet-induced obese mice.

Type 2 diabetes (T2D) is a metabolic disease affecting >370 million people worldwide. It is characterized by obesity-induced insulin resistance, and growing evidence has indicated that this causative link between obesity and insulin resistance is associated with visceral adipose tissue inflammation. However, using anti-inflammatory drugs to treat insulin resistance and T2D is not a common practice. We recently applied a bioinformatics methodology to open public data and found that CD44 plays a critical role in the development of adipose tissue inflammation and insulin resistance. In this report, we examined the role of CD44 in T2D by administering daily injections of anti-CD44 monoclonal antibody (mAb) in a high-fat-diet mouse model. Four weeks of therapy with CD44 mAb suppressed visceral adipose tissue inflammation compared with controls and reduced fasting blood glucose levels, weight gain, liver steatosis, and insulin resistance to levels comparable to or better than therapy with the drugs metformin and pioglitazone. These findings suggest that CD44 mAb may be useful as a prototype drug for therapy of T2D by breaking the links between obesity and insulin resistance.

Relationship between Mutations of the Pectin Methylesterase Gene in Soybean and the Hardness of Cooked Beans.

Hardness of cooked soybeans [Glycine max (L). Merr.] is an important attribute in food processing. We found one candidate gene, Glyma03g03360, to be associated with the hardness of cotyledons of cooked soybeans, based on a quantitative trait locus and fine-scale mapping analyses using a recombinant inbred line population developed from a cross between two Japanese cultivars, "Natto-shoryu" and "Hyoukei-kuro 3". Analysis of the DNA sequence of Glyma03g03360, a pectin methylesterase gene homologue, revealed three patterns of mutations, two of which result in truncated proteins and one of which results in an amino acid substitution. The truncated proteins are presumed to lack the enzymatic activity of Glyma03g03360. We classified 24 cultivars into four groups based on the sequence of Glyma03g03360. The texture analysis using the 22 cultivars grown in different locations indicated that protein truncation of Glyma03g03360 resulted in softer cotyledons of cooked soybeans, which was further confirmed by texture analysis performed using F2 populations of a cross between "Enrei" and "LD00-3309", and between "Satonohohoemi" and "Sakukei 98". A positive correlation between hardness and calcium content implies the possible effect of calcium binding to pectins on the hardness of cooked soybean cotyledons.

Differential induction of serum interleukin-6 and -12 by interferon-alpha and -beta administration in chronic hepatitis C patients.

Interferon (IFN) is widely used to manage chronic hepatitis C virus (HCV) infections. It contributes to the production of various cytokines, and alters cytokine interactions in vivo. Among them, interleukin (IL)-12, is thought to shift the immune system to Th-1 dominance and to, therefore, have a beneficial effect on eradication of HCV. On the other hand, IL-6 seems to be related to the acute inflammatory phenomenon, such as fever accompanied with IFN therapy. We studied the time courses of the serum IL-12 and IL-6 levels of patients with HCV infections treated with either IFN-alpha or IFN-beta. On the first day, both IFN-alpha and IFN-beta significantly induced serum IL-6, but when the administration was repeated, the serum IL-6 levels gradually decreased in both the IFN-alpha and the IFN-beta groups. The serum IL-6 levels correlated well with the daily peak body temperature of the patients, which also went down with repeated IFN administration. Serum IL-12 production was induced by IFN-alpha not by IFN-beta. The induced IL-12 decreased to baseline by repeating IFN-alpha administration. These results suggested that IFN-alpha and IFN-beta induce cytokine production in a different manner, and that their effect on cytokine interactions is short-term and reduced by repeated administration. Therefore, the induction of serum IL-12 production by IFN did not seem to have a critical effect during the early stage of IFN therapy.