5′-Phosphodiesterase Formation by Cultured Plant Cells

5′-Phosphodiesterase (5′-PDase) which degrades RNA to nucleoside-5′-monophosphates was investigated in various kinds of plant calli, and the calli of Vinca rosea and Phytolacca americana were found to have the high activity. The liquid culture conditions of the cells of V. rosea were examined. Three mg of kinetin and 0.5 mg of 2,4-dichlorophenoxyacetic acid per liter in the Murashige and Skoog medium were optimal for the growth and the 5′-PDase formation. Under the optimal conditions, time courses of the cell growth and the enzyme formation were measured.

The 5′-PDase of the cultured cells of V. rosea in suspension showed the maximal activity at pH 8.0 and 60°C. A comparison of 5′-PDase of the cultured cells and of the mother plant of V. rosea was carried out and it was found that the cultured cells had more than 30 times as much 5′-PDase activity as the mother plant on dry cell weight basis.     

Involvement of Fyn tyrosine kinase in actin stress fiber formation in fibroblasts

Lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) activated Fyn tyrosine kinase and induced stress fiber formation, which was blocked by pharmacological inhibition of Fyn, gene silencing of Fyn, or dominant negative Fyn. Overexpressed constitutively active Fyn localized at both ends of F-actin bundles and triggered stress fiber formation, only the latter of which was abolished by Rho-kinase (ROCK) inhibition. SPC, but not LPA, induced filopodia-like protrusion formation, which was not mediated by Fyn and ROCK. Thus, Fyn appears to act downstream of LPA and SPC to specifically stimulate stress fiber formation mediated by ROCK in fibroblasts.     

Integrin-dependent cell behavior on ECM peptide-conjugated chitosan membranes

Extracellular matrix (ECM) plays an important role in tissue regeneration by promoting cell adhesion, migration, proliferation, and differentiation. ECM mimetics are of importance for tissue engineering because of their functions as scaffolds for cells. Previously, we developed bioactive laminin-derived peptide-conjugated chitosan membranes and demonstrated their cell- and peptide-type specific functions. Here, we conjugated twelve integrin-binding peptides derived from ECM proteins onto chitosan membranes and examined biological activity. Seven peptide-chitosan membranes promoted human foreskin fibroblast attachment. Additionally, FIB1 (YAVTGRGDSPAS; from fibronectin), A99 (AGTFALRGDNPQG; from laminin alpha1 chain), EF1zz (ATLQLQEGRLHFXFDLGKGR, X = Nle; from laminin alpha1 chain), and 531 (GEFYFDLRLKGDKY; from collagen alpha1 (IV) chain) conjugated chitosan membranes promoted integrin-dependent cell adhesion. Various integrins, including alphav, beta1, and beta3, were involved in the cell adhesion to the peptide-chitosan membranes. Further, only the FIB1- and A99-chitosan membranes promoted neurite outgrowth with PC12 rat pheochromocytoma cells. These data demonstrate that peptide-chitosan membranes can regulate specific integrin-mediated cell responses and are useful constructs as ECM mimetics.     

Soluble Lutheran/basal cell adhesion molecule is detectable in plasma of hepatocellular carcinoma patients and modulates cellular interaction with laminin-511 in vitro

Lutheran (Lu), an immunoglobulin superfamily transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a subunit of laminin-511 (LM-511) that is a major component of basement membranes in various tissues. Our previous study showed that Lu/B-CAM was cleaved by MT1-MMP and released from cell surfaces. In this study we examined the soluble Lu/B-CAM in culture media and in plasma of mice bearing HuH-7 hepatocellular carcinoma (HCC) cells and patients with HCC. Two HCC cell lines, HepG2 and HuH-7, released Lu/B-CAM into the culture media. Although Lu/B-CAM was cleaved by MT1-MMP in HuH-7 cells, HepG2 cells released Lu/B-CAM in a MMP-independent manner. The concentration of Lu/B-CAM released into mouse plasma correlated with tumor size. Moreover the soluble Lu/B-CAM in plasma of HCC patients was significantly decreased after resection of the tumor. Immunohistochemical studies showed that although the expression of Lu/B-CAM was observed in most HCCs, MT1-MMP was not always expressed in tumor tissues, suggesting that a part of Lu/B-CAM in plasma of HCC patients was also released in a MMP-independent manner. In vitro studies showed that the soluble Lu/B-CAM released from HCC cells bound to LM-511. Moreover the soluble Lu/B-CAM influenced cell migration on LM-511. These results suggest that soluble Lu/B-CAM serves as not only a novel marker for HCC but also a modulator in tumor progression.     

Nrf2 Enhances Cholangiocyte Expansion in Pten-Deficient Livers

Keap1-Nrf2 system plays a central role in the stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 activity is abrogated in response to oxidative or electrophilic stresses, leading to Nrf2 stabilization and coordinated activation of cytoprotective genes. We recently found that nuclear accumulation of Nrf2 is significantly increased by simultaneous deletion of Pten and Keap1, resulting in the stronger activation of Nrf2 target genes. To clarify the impact of the cross talk between the Keap1-Nrf2 and Pten–phosphatidylinositide 3-kinase–Akt pathways on the liver pathophysiology, in this study we have conducted closer analysis of liver-specific Pten::Keap1 double-mutant mice (Pten::Keap1-Alb mice). The Pten::Keap1-Alb mice were lethal by 1 month after birth and displayed severe hepatomegaly with abnormal expansion of ductal structures comprising cholangiocytes in a Nrf2-dependent manner. Long-term observation of Pten::Keap1-Alb::Nrf2^+/− mice revealed that the Nrf2-heterozygous mice survived beyond 1 month but developed polycystic liver fibrosis by 6 months. Gsk3 directing the Keap1-independent degradation of Nrf2 was heavily phosphorylated and consequently inactivated by the double deletion of Pten and Keap1 genes. Thus, liver-specific disruption of Keap1 and Pten augments Nrf2 activity through inactivation of Keap1-dependent and -independent degradation of Nrf2 and establishes the Nrf2-dependent molecular network promoting the hepatomegaly and cholangiocyte expansion.     

Diel changes of temperature in the nests of two Polybia species, P. paulista and P. occidentalis (Hymenoptera, Vespidae) in the subtropical climate

Temperatures in two mature Polybia nests were measured to study the thermal conditions of nests. Temperatures in a Polybia paulista nest were measured when it had adult wasps (trial 1) and after the adult wasps were removed (trial 2), and temperatures in a P. occidentalis nest were measured when inhabited by adult wasps (trial 3). In each trial, C–C thermocouples were set at four points in the nest and at two points outside to discover the ambient and substratum temperatures. In trial 1, nest temperatures basically followed the ambient temperature, but during the day the nest temperatures were lower than the ambient temperature and this relationship was reversed during the night. The temperature fluctuation of the inner substratum points was smaller than that of the outer points. The trend of trial 2 was closely similar to that of trial 1, showing a virtual lack of thermal effects from the presence of adult wasps. In trial 3, the changing pattern in the P. occidentalis nest showed a trend similar to that in the P. paulista nest, but the inside temperatures rose quickly when the nest received direct sunlight in the morning. From these results, thermal characteristics in the Polybia nests are discussed in relation to the nest architecture and their geographical distribution.     

Induction of estrus in pubertal miniature gilts

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.     

Analysis of proteolytic processing during specific antigen presentation

In this report we have studied the effect of protease inhibitors on B-cell-antigen processing. As a source of antigen-presenting B cells we have utilized transformants transfected with a vector carrying immunoglobulin (Ig) genes specific for the hapten trinitrophenyl (TNP). B-cell-specific (TNP-proteins) and nonspecific antigen-presentation activities were blocked to the same extent upon addition of inhibitors for protease and endosomal function. Interestingly, the effect of leupeptin, a thiol protease inhibitor, varied depending on the antigen and helper T cells utilized. These results suggest that specific groups of proteases may be required for antigen processing so that discrete antigenic epitopes in association with major histocompatibility complex molecules can be recognized by interacting T cells.