Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.     

Structural requirement of tunicamycin V for MraY inhibition

Elucidating a structure-activity relationship study by evaluating a series of truncated analogues is a simple but important and effective tactic in medicinal chemistry based on natural products with a large and complex chemical structure. In this study, a series of truncated analogues of tunicamycin V were designed and synthesized and their MraY inhibitory activity was investigated in order to gain insight into the effect of these moieties on MraY inhibition.     

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.