Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.     

An androgen-dependent pilosebaceous tumor spontaneously developed in the Japanese house musk shrew Suncus murinus

Following administration of [3H]testosterone to castrated male Japanese house musk shrews (Suncus murinus), radioactive metabolites were detected in sidegland nuclei and the major one of them was dihydrotestosterone (DHT). The androgen binding capacity of the cytoplasmic fraction of sideglands was measured in vitro by the use of [3H]R1881 as a ligand. The binding showed a high affinity for R1881 (Kd = 6.2 X 10(-10) M) and a low capacity (Bmax = 22 fmol/mg protein). Sucrose density gradient centrifugation brought about a peak of [3H]R1881 in the 7S region in low ionic strength buffer. Their characteristics as described above are consistent with those of other androgen target organs. A cutaneous pilosebaceous tumor, which spontaneously developed on the sidegland of old male S. murinus, was transplanted to nude athymic mice. It grew in males only and failed to grow in females and castrated males. A specific androgen binding was found in this tumor (Kd = 7.8 X 10(-10) M, Bmax = 100 fmol/mg protein). Therefore, this transplantable pilosebaceous tumor is androgen-dependent and can be utilized as a new suitable model in the study of the mechanism of androgen on tumor development.     

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β-1, β-2, β-3, β-4 and β-5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.     

Simple dehydration treatment promotes plantlet regeneration of rice (Oryza sativa L.) callus

Summary To increase plantlet regeneration frequency, rice callus was dehydrated in a Petri dish with a single layer of filter paper prior to transfer to the regeneration medium. With a 24 h dehydration treatment, the regeneration frequency was increased to 47 %, while the regeneration frequency of the untreated control was less than 5 %. This relatively simple method provides an alternative method for improving the regeneration frequency of rice callus.     

Effect of solar near ultraviolet radiation on growth of Chlorella cells

Cells of Chlorella regularis (Artari) Oltmanns (S-50) were grown under solar radiation in Tokyo, using a newly constructed outdoor culture system. The maximum specific growth rate (log₂ unit h⁻¹) was about 0.3, which was a little lower than the highest value reported using artificial light in the laboratory. The near ultraviolet light, consisting mostly of UV-A, inhibited the growth from 10 to 40% in summer mornings; the inhibition was not significant in the afternoon. The percentage inhibition was correlated with the total dose of the ultraviolet radiation.     

The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process

Several lines of in vitro evidence suggest the potential role of IFN-gamma in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-gamma in the skin wound healing process utilizing WT and IFN-gamma KO mice. In WT mice, excisional wounding induced IFN-gamma mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-gamma KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-gamma KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-gamma KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-beta1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-gamma augmented the TGF-beta1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-gamma/Stat1 and TGF-beta1/Smad signaling pathways in the wound healing process.     

Activation mechanism of c-Jun amino-terminal kinase in the course of neural differentiation of P19 embryonic carcinoma cells

P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cepsilon, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.     

Synthetic seeds as an application of mass production of somatic embryos

Production of encapsulatable unit, scale-up of the production system, and encapsulation technologies were developed as an integrated technology for transplant production using somatic embryos of celery and carrot. Encapsulatable units, somatic embryos improved in their quality for encapsulation, showed 80% conversion without encapsulation and any nutrient supply on a nursery medium in a greenhouse. The high conversion ability was also shown by carrot encapsulatable unit produced with a newly developed 8-liter culture jar, where 5.09×10⁵ torpedo stage embryos were produced and 4.10×10⁴ encapsulatable units were obtained from them.A novel self-breaking gel beads and sustained release microcapsule as an artificial endosperm were developed for the encapsulation of carrot embryos. Also a whole production system for synthetic seed was developed with which 80 thousand beads could be produced per day. The encapsulated carrot embryos showed 52% conversion frequency after sowing on a humid soil in greenhouse.Abbreviations BA benzyladenine - GA gibberellic acid - MC microcapsule