Successful long-term preservation of abscisic-acid-treated and desiccated carrot somatic embryos

We have previously reported that strong desiccation tolerance in carrot somatic embryos can be achieved by treatment with abscisic acid (ABA). In this study, we examined the possibility of long-term preservation of ABA-treated and desiccated somatic embryos. Somatic embryos that had been desiccated after treatment with ABA survived for at least 169 weeks at –25 °C. By contrast, somatic embryos that had not been desiccated after treatment with ABA survived for at least 24 weeks at +5 °C but died at –25 °C. Received: 11 July 1998 / Revision received: 20 October 1998 / Accepted: 20 November 1998     

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.     

Ammonia detoxification by accelerated oxidation of branched chain amino acids in brains of acute hepatic failure rats

BCAA aminotransferase and BCKA dehydrogenase activities are increased in the mitochondrial fractions from the brains of hepatic failure rats treated with two-thirds removal of CCl4-injured liver. Cerebral leucine decarboxylation was accelerated, and it well correlated with arterial blood ammonia levels. Elevation of brain ammonia content following an intraperitoneal injection of ammonium acetate to hepatic failure rats could be prevented by intravenous infusion of BCAA. Significantly increased brain glutamic acid, glutamine, and alanine contents were noted. These results suggested that accelerated brain BCAA catabolism in acute hepatic failure rats reduce the neurotoxicity of ammonia by promoting the synthesis of glutamic acid and glutamine from BCAA.     

The critical role of the stem region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyltransferase V. The involvement of a domain homophilic interaction

We demonstrated that a region in the stem of N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident protein, is not required for enzyme activity but serves as functional domain, responsible for intracellular localization. Deletion of the domain led to complete retention of the kinetic properties but resulted in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle. Furthermore, it was found that the presence of this domain is exclusively responsible for homo-oligomer formation. This homophilic interaction appears to involve a hydrophobic cluster of residues in the alpha-helix of the domain, as indicated by secondary structure predictions. These findings suggest that the domain specifically participates in the Golgi retention of GnT-V, probably via inducing homo-oligomer formation, and would also provide a possible mechanism for the oligomerization, which is critical for localization in the Golgi.     

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.     

Association of single-nucleotide polymorphisms in the suppressor of cytokine signaling 2 (SOCS2) gene with type 2 diabetes in the Japanese

Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.     

Sequential changes in genome-wide DNA methylation status during adipocyte differentiation

DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. To determine the epigenetic changes during adipocyte differentiation, we investigated the sequential changes in DNA methylation status of 3T3-L1 cells at the growing, confluent, postconfluent and mature adipocyte cell stages. Treatment of 3T3-L1 cells with 5-aza-2′-deoxycytidine inhibited differentiation in a stage-dependent manner, supporting the idea that formation of accurate DNA methylation profile, consisting of methylated and unmethylated T-DMRs, may be involved in differentiation. Analysis by methylation-sensitive quantitative real-time PCR of the 65 known T-DMRs which contain NotI sites detected 8 methylations that changed during differentiation, and the changes in the patterns of these methylations were diverse, confirming that the differentiation process involves epigenetic alteration at the T-DMRs. Intriguingly, the dynamics of the methylation change vary depending on the T-DMRs and differentiation stages. Restriction landmark genomic scanning detected 32 novel T-DMRs, demonstrating that differentiation of 3T3-L1 cells involves genome-wide epigenetic changes by temporal methylation/demethylation, in addition to maintenance of a static methylated/demethylated state, and both depend on differentiation stage.     

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1^−/−-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.Subject terms: Stress signalling, Cell death, Inflammation