GRAS transcription factors regulate cell division planes in moss overriding the default rule.

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

L-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic ß-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in L-cysteine-treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.

Depth of Interbreed Difference in Postmortem Bovine Muscle Determined by CE-FT/MS and LC-FT/MS Metabolomics.

Japanese Brown (JBR) cattle have moderately marbled beef compared to the highly marbled beef of Japanese Black (JBL) cattle; however, their skeletal muscle properties remain poorly characterized. To unveil interbreed metabolic differences over the previous results, we explored the metabolome network changes before and after postmortem 7-day aging in the trapezius muscle of the two cattle breeds by employing a deep and high-coverage metabolomics approach. Using both capillary electrophoresis (CE) and ultra-high-performance liquid chromatography (UHPLC)-Fourier transform mass spectrometry (FT/MS), we detected 522 and 384 annotated peaks, respectively, across all muscle samples. The CE-based results showed that the cattle were clearly separated by breed and postmortem age in multivariate analyses. The metabolism related to glutathione, glycolysis, vitamin K, taurine, and arachidonic acid was enriched with differentially abundant metabolites in aged muscles, in addition to amino acid (AA) metabolisms. The LC-based results showed that the levels of bile-acid-related metabolites, such as tauroursodeoxycholic acid (TUDCA), were high in fresh JBR muscle and that acylcarnitines were enriched in aged JBR muscle, compared to JBL muscle. Postmortem aging resulted in an increase in fatty acids and a decrease in acylcarnitine in the muscles of both cattle breeds. In addition, metabolite set enrichment analysis revealed that JBR muscle was distinctive in metabolisms related to pyruvate, glycerolipid, cardiolipin, and mitochondrial energy production, whereas the metabolisms related to phosphatidylethanolamine, nucleotide triphosphate, and AAs were characteristic of JBL. This suggests that the interbreed differences in postmortem trapezius muscle are associated with carnitine/acylcarnitine transport, ß-oxidation, tricarboxylic acid cycle, and mitochondrial membrane stability, in addition to energy substrate and AA metabolisms. These interbreed differences may characterize beef quality traits such as the flavor intensity and oxidative stability.

Unmasking of LPA1 receptor-mediated migration response to lysophosphatidic acid by interleukin-1ß-induced attenuation of Rho signaling pathways in rat astrocytes.

Action mechanism of lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), and lysophosphatidic acid (LPA) to regulate motility, an important process of astrogliosis, was investigated in rat astrocytes. While LPA exerted no significant effect on the cell migration, the prior treatment of the cells with LPS or IL-1ß resulted in the appearance of migration activity in response to LPA. The LPS induction of the migration response to LPA was associated with the production of IL-1ß precursor protein and inhibited by the IL-1 receptor antagonist. The IL-1ß treatment also allowed LPA to activate Rac1. The LPA-induced Rac1 activation and migration were inhibited by pertussis toxin, a small interfering RNA specific to LPA(1) receptors, and LPA(1) receptor antagonists, including Ki16425. However, the IL-1ß treatment had no appreciable effect on LPA(1) receptor mRNA expression and LPA-induced activation of ERK, Akt, and proliferation. The induction of the migration response to LPA by IL-1ß was inhibited by a constitutively active RhoA. Moreover, LPA significantly activated RhoA through the LPA(1) receptor in the control cells but not in the IL-1ß-treated cells. These results suggest that IL-1ß inhibits the LPA(1) receptor-mediated Rho signaling through the IL-1 receptor, thereby disclosing the LPA(1) receptor-mediated G(i) protein/Rac/migration pathway.

Surveillance of Shiga toxin-producing Escherichia coli and Campylobacter spp. in wild Japanese deer (Cervus nippon) and boar (Sus scrofa).

Increasing game meat consumption in Japan requires the dissemination of safety information regarding the presence of human pathogens in game animals. Health information regarding the suitability of these animals as a meat source is not widely available. In this study, we aimed to evaluate the safety of game meat and detect potential human pathogens in wild deer (Cervus nippon) and boar (Sus scrofa) in Japan. Fecal samples from 305 wild deer and 248 boars of Yamaguchi, Kagoshima, and Tochigi prefectures collected monthly for 2 years were examined for the prevalence of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter spp. STEC was isolated from 51 deer consistently throughout the year and from three boars; O-antigen genotype O146, the expression of stx2b, and eaeA absence (n=33) were the major characteristics of our STEC isolates. Other serotypes included the medically important O157, stx2b or stx2c, and eaeA-positive (n=4) and O26, stx1a, and eaeA-positive strains (n=1). Campylobacter spp. were isolated from 17 deer and 31 boars. Campylobacter hyointestinalis was the most common species isolated from 17 deer and 25 boars, whereas Campylobacter lanienae and Campylobacter coli were isolated from three and two boars, respectively. Seasonal trends for the isolation of these bacteria were not significant. This study demonstrates that wild game animals carry human pathogens; therefore, detailed knowledge of the safe handling of game meat is needed to prevent foodborne infections.

Characterizing key nucleotide polymorphisms of hepatitis C virus-disease associations via mass-spectrometric genotyping.

As more than 80% of hepatocellular carcinoma patients in Japan also suffer from hepatitis C virus infections some time in their medical history, identifying genetic aberrations associated to hepatitis C virulence in these individuals remains a high priority in the diagnosis and treatment of hepatocellular carcinoma. From the BioBank Japan Project, we acquired 480 subjects of hepatocellular carcinoma, chronic hepatitis and liver cirrhosis, and genotyped 131 clinically relevant host single nucleotide polymorphisms to survey the potential association between certain risk alleles and genes to a patient's predisposition to hepatitis C and liver cancer. Among those polymorphisms, we found 12 candidates with statistical significance to support association with hepatitis C virus susceptibility and genetic predisposition to hepatocellular carcinoma. SNPs in genes such as XPC, FANCA, KDR and BRCA2 also suggested likely connections between hepatitis C virus susceptibility and the contraction of liver diseases. Single nucleotide polymorphisms reported here provided suggestions for genes as biomarkers and elucidated insights briefing the linkage of hepatitis C virulence to the alteration of healthy liver genomic landscape as well as liver disease progression.

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes.

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1-/- mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.

Pyruvate kinase M1 interacts with A-Raf and inhibits endoplasmic reticulum stress-induced apoptosis by activating MEK1/ERK pathway in mouse insulinoma cells.

Apoptotic death of pancreatic ß cells is a major cause of type 2 diabetes mellitus (T2D) progression. Two isoforms of pyruvate kinase, PKM1 and PKM2, have been reported to participate in cell death in several cell types; however, little is known about their causal pathways in pancreatic ß-cell death. We examined whether the suppression of PKM1 or PKM2 affects endoplasmic reticulum (ER) stress-induced apoptosis in a pancreatic ß-cell line, MIN6, and Beta-TC-6 and found that knockdown of PKM1, but not of PKM2, leads to the induction of ER stress-induced apoptosis in these cells. We also investigated the mechanism by which PKM1 inhibits ER stress-induced apoptosis. We confirmed that PKM1 interacts with A-Raf, an upstream regulator of the MEK/ERK pathway, and that this interaction contributes to MEK1 phosphorylation by A-Raf. PKM1 knockdown suppresses the phosphorylation of MEK, ERK, and caspase-9 (Thr125), which is phosphorylated by the MEK/ERK pathway, thereby inhibiting the cleavage and activation of caspase-9. Thus, PKM1 knockdown activates the caspase-9/caspase-3 pathway under ER stress conditions and leads to apoptosis.

Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response.

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells.

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P(1) to inhibition through S1P(2). LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA(1) receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominant-negative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways are equally important for LPA(1) receptor-mediated migration.

HDL-like lipoproteins in cerebrospinal fluid affect neural cell activity through lipoprotein-associated sphingosine 1-phosphate.

High-density lipoprotein (HDL)-associated sphingosine 1-phosphate mediates a variety of lipoprotein-induced actions in vascular cell systems. However, it remains unknown whether extracellular S1P is associated with lipoproteins to exert biological actions in central nervous system. Human cerebrospinal fluid (CSF) induced rat astrocyte migration in a manner sensitive to S1P receptor antagonist VPC23019 and the migration activity was recovered in S1P fraction by thin-layer chromatography. Density-gradient separation of CSF revealed that the major S1P activity was detected in the HDL fraction. In conditioned medium of rat astrocytes cultured with sphingosine, the S1P activity was recovered again in the HDL fraction. The HDL fraction also induced migration of astrocytes and process retraction of oligodendrocytes in a manner similar to S1P. We concluded that S1P is accumulated in HDL-like lipoproteins in CSF and mediates some of lipoprotein-induced neural cell functions in central nervous system.