Identification of myosin II as a binding protein to the PH domain of protein kinase B

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.     

Contamination of public facilities with Dermatophagoides mites (Acari: Phyroglyphidae) in Japan

The distribution of allergenic mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus, was surveyed among public facilities in Japan, in order to assess the risk of people, including allergic patients, being exposed to mite allergens outside homes. Dust samples were collected using a vacuum cleaner from bedclothes, floors and other materials in four hospitals, two hotels, two ryokans, one film theatre and four office buildings. Fine dust fractions of these samples were examined for mite antigens by enzyme-linked immunosorbent assay. Overall, clinically important antigen levels of 50 g m–2 (corresponding to approximately 500 mites m–2) or more were detected in 71% of samples from chairs in a film theatre and 42% of samples from tatami and carpet floors of ryokans, whereas such serious mite contamination was not observed in samples from hospitals, hotels and offices. High mean antigen levels were observed in chairs in a film theatre (98.2 g m–2) and floors of ryokans (81.4 g m–2), whereas samples from other facilities showed mean antigen levels of <11 g m–2. Hospitals had individual antigen levels of 50 g m–2 in 2% of mattresses and 5% of painted and tiled floors but not in comforters, futons and pillows. No samples obtained from bedclothes in hotels and ryokans had antigen levels of 50 g m–2. In office buildings where the air was fully controlled by a central air-conditioning system all the year round, 99% of samples showed low antigen levels of <10 g m–2. © Rapid Science Ltd. 1998     

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.     

Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.     

Distribution of minerals in quinoa (Chenopodium quinoa Willd.) seeds

The distribution of minerals in quinoa (Chenopodium quinoa Willd.) seed was examined using energy dispersive X-ray microanalysis (EDX) in combination with scanning electron microscopy (SEM). Phosphorus, K, and Mg coincided in localization in embryonic tissue. Since phytin globoids have been known to localize in protein bodies in embryonic cells of quinoa seed, it is thought that P is attributed to phytic acid and that K and Mg form to phytate. Calcium and K were present in the pericarp, where the cell wall is thickly developed, suggesting that these minerals are associated with pectin. Sulfur occurred in embryonic tissues, which would be derived from sulfur amino acid residues of storage proteins concentrated in the tissues. Abrasion of quinoa seeds resulted particularly in decrease in Ca content.     

A bitter melon extract inhibits the P-glycoprotein activity in intestinal Caco-2 cells: monoglyceride as an active compound

P-glycoprotein (P-gp) is a 170 kDa membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily. In normal tissues, P-gp functions as an ATP-dependent efflux pump that excretes highly hydrophobic xenobiotic compounds, playing an important role in protecting the cells/tissues from xenobiotics. In the present study, chemical substances that could directly modulate the intestinal P-gp activity were searched in vegetables and fruits. By using human intestinal epithelial Caco-2 cells as a model of the small intestinal cells, we observed that a bitter melon fraction extracted from 40% methanol showed the greatest increase of the rhodamine-123 accumulation by Caco-2 cells. Inhibitory compounds in the bitter melon fraction were then isolated by HPLC using Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. It is interesting that certain types of monoglyceride might be involved in the drug bioavailability by specifically inhibiting the efflux mediated by P-gp.     

Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.     

Evidence that sucrose loaded into the phloem of a poplar leaf is used directly by sucrose synthase associated with various beta-glucan synthases in the stem

Sucrose (Suc) synthase (SuSy) is believed to function in channeling UDP-Glc from Suc to various beta-glucan synthases. We produced transgenic poplars (Populus alba) overexpressing a mutant form (S11E) of mung bean (Vigna radiata) SuSy, which appeared in part in the microsomal membranes of the stems. Expression of SuSy in these membranes enhanced the incorporation of radioactive Suc into cellulose, together with the metabolic recycling of fructose (Fru), when dual-labeled Suc was fed directly into the phloem of the leaf. This overexpression also enhanced the direct incorporation of the glucosyl moiety of Suc into the glucan backbone of xyloglucan and increased recycling of Fru, although the Fru recycling system for cellulose synthesis at the plasma membrane might differ from that for xyloglucan synthesis in the Golgi network. These findings suggest that some of the Suc loaded into the phloem of a poplar leaf is used directly by SuSys associated with xyloglucan and cellulose synthases in the stem. This may be a key function of SuSy because the high-energy bond between the Glc and Fru moieties of Suc is conserved and used for polysaccharide syntheses in this sink tissue.     

Histopathology, pathogenesis and molecular genetics in primary central nervous system lymphomas

Recent increases in the incidence of primary central nervous system lymphoma (PCNSL), a rare non-Hodgkin's lymphoma arising in the brain, have been noted in both immunodeficient and immunocompetent patients. Compared with lymphomas originating outside the central nervous system, the biology of PCNSL at the molecular or cytogenetic level has not been well characterized, yet it is important to thoroughly understand the etiology of this rare malignant lymphoma if effective therapies are to be developed. This review will focus on the epidemiology, clinical aspects, histopathology, pathogenesis, and molecular genetics of this aggressive, extranodal lymphoma in immunocompetent patients.