The carboxyl terminal sequence of nucleolar protein B23.1 is important in its DNA polymerase alpha-stimulatory activity

The protein B23 is a major nucleolar phosphoprotein comprising two isoforms, B23.1 and B23.2, which differ only in their carboxyl-terminal short sequences, the N-terminal 255 residues being identical in both forms. Both B23.1 and B23.2 stimulated immunoaffinity-purified calf thymus DNA polymerase alpha in a dose-dependent manner. The stimulatory effect of protein B23.1, the longer isoform, was found to be 2-fold greater than that of B23.2. Purified DNA polymerase alpha bound tightly to a protein B23.1-immobilized column, while it bound weakly to a protein B23.2-immobilized column. Surface plasmon resonance studies by BIAcore further showed that protein B23.1 bound to the DNA polymerase alpha-(dA).(dT) complex more tightly than did protein B23.2. The protein B23 isoforms appear to interact directly with the DNA polymerase alpha protein and not through the bound nucleic acid. These observations indicated that protein B23 physically bound to the DNA polymerase alpha and stimulated the enzyme activity. Product analyses showed that protein B23 greatly enhanced the reaction both in amount and length of product DNA, whereas it did not significantly alter the processivity of polymerization. In contrast, protein B23 effectively protected DNA polymerase alpha from heat inactivation. These results suggest that protein B23 stabilizes DNA polymerase alpha that is detached from product DNA, allowing the enzyme to be recruited for further elongation. Moreover, experiments using various C-terminal deletion mutants of protein B23 indicated that 12 amino acids at the C-terminal end of B23.1, which are absent in B23.2, may be essential for the full stimulation of the DNA polymerase alpha.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.     

Enzymatic synthesis of aliphatic beta-lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase

Aliphatic beta-lactosides were directly synthesized by beta-lactosyl transfer reaction from p-nitrophenyl beta-lactoside (Lac beta-pNP) to various 1-alkanols (n = 2-12), utilizing commercially available cellulase preparation of Trichoderma reesei C1. With ethanol acceptor, the enzyme induced ethyl beta-lactoside (1) in 18% yield based on the donor added in aqueous buffer system. When 1-octanol and dodecanol were acceptors, octyl beta-lactoside (2) and dodecyl beta-lactoside (3) were also obtained as transfer products, respectively. In both cases, the addition of sodium cholate as detergent to the reaction system ensured a sufficient solubility of these acceptors and resulted in a remarkable increase of the desired compounds (5-13% yields based on the donor added). Furthermore, the enzyme catalyzed the N-acetyllactosaminyl transfer reaction from p-nitrophenyl beta-N-acetyllactosaminide (LacNAc beta-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides, Gal beta1-4GlcNAc beta1-4Man (4) and Gal beta1-4GlcNAc beta1-4Glc (5), respectively. The enzyme activities transferring lactosyl and N-acetyllactosaminyl groups were not separated by chromatographies using DEAE-Sepharose Fast Flow and Sephadex 75 pg columns, indicating that the two reactions were catalyzed by a single enzyme. It was specified that a single enzyme works both transglycosylations, based on the substrate competition assay on hydrolysis.     

Facile enzymatic conversion of lactose into lacto-N-tetraose and lacto-N-neotetraose

Lacto-N-tetraose (Galbeta1 -3GlcNAcbeta1-3Galbeta1-4Glc, LNT) and lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, LNnT) were enzymatically synthesized by consecutive additions of GlcNAc and Gal residues to lactose. Lacto-N-triose II (GlcNAcbeta1-3Galbeta1-4Glc) was prepared first by the transfer of GlcNAc from UDP-GlcNAc to lactose by beta-1,3-N-acetylglucosaminyltransferase from bovine serum. The resulting lacto-N-triose II was converted into LNT and LNnT utilizing two kinds of beta-D-galactosidase-mediated transglycosylations. Thus, beta-D-galactosidase from Bacillus circulans ATCC31382 induced regioselective galactosyl transfer from o-nitrophenyl beta-D-galactoside to the OH-3" position of lacto-N-triose II, and commercially available beta-D-galactosidase from B. circulans to the OH-4" position of lacto-N-triose II. These convenient processes are suitable for large-scale preparations of LNT and LNnT. As another method, LNT was directly synthesized from lactose as an initial substance, utilizing lacto-N-biosidase (Aureobacterium sp. L-101)-mediated transglycosylation with Galbeta1-3GlcNAcbeta-pNP donor.     

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.     

Photoperiod- and temperature-dependent regulation of pupal beige/black polymorphism in the small copper butterfly, Lycaena phlaeas daimio Seitz

The small copper butterfly, Lycaena phlaeas daimio, has pupal beige/black polymorphism, the development of which is found to be controlled in an apparent association with the development of adult seasonal polymorphism (spring and summer morphs) by photoperiod and temperature in the larval stages. That is, the pupae of beige and black types developed under long-day and short-day conditions tend to develop into brown-winged and red-winged adults, respectively. In addition, a large proportion of long-day pharate pupae chilled at 4 degrees C for 5 days were observed to develop into pupae whose head-thoracic complexes and abdomens were judged to be of the black and intermediate types, respectively. They developed into adults with redder wings as compared to those obtained from unchilled pupae. The results indicate that the physiological mechanism underlying the photoperiodic control of the development of adult seasonal polymorphism may also play a significant role in the determination of pupal beige/black polymorphism in L. phlaeas daimio. Furthermore, cuticle melanization was found to be induced in the head-thoracic complexes of pupae by chilling of the pharate pupae. Melanization of pupal cuticle seems to occur in a close association with the development of reddish-winged adults.     

Cytotoxicity and oxidative stress induced by the glyceraldehyde-related Maillard reaction products for HL-60 cells

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nalpha-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress.We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nalpha-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930-932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production.This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.     

Tomato paste fraction inhibiting the formation of advanced glycation end-products

A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.     

Establishment and characterization of a new human glioblastoma cells line, NYGM

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.     

Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.