Effect of intraportal glucagon-like peptide-1 on glucose metabolism in conscious dogs

Arteriovenous difference and tracer ([3-(3)H]glucose) techniques were used in 42-h-fasted conscious dogs to identify any insulin-like effects of intraportally administered glucagon-like peptide 1-(7-36)amide (GLP-1). Each study consisted of an equilibration, a basal, and three 90-min test periods (P1, P2, and P3) during which somatostatin, intraportal insulin (3-fold basal) and glucagon (basal), and peripheral glucose were infused. Saline was infused intraportally in P1. During P2 and P3, GLP-1 was infused intraportally at 0.9 and 5.1 pmol. kg(-1). min(-1) in eight dogs, at 10 and 20 pmol. kg(-1). min(-1) in seven dogs, and at 0 pmol. kg(-1). min(-1) in eight dogs (control group). Net hepatic glucose uptake was significantly enhanced during GLP-1 infusion at 20 pmol. kg(-1). min(-1) [21.8 vs. 13.4 micromol. kg(-1). min(-1) (control), P < 0.05]. Glucose utilization was significantly increased during infusion at 10 and 20 pmol. kg(-1). min(-1) [87.3 +/- 8.3 and 105.3 +/- 12.8, respectively, vs. 62.2 +/- 5.3 and 74.7 +/- 7.4 micromol. kg(-1). min(-1) (control), P < 0.05]. The glucose infusion rate required to maintain hyperglycemia was increased (P < 0.05) during infusion of GLP-1 at 5.1, 10, and 20 pmol. kg(-1). min(-1) (22, 36, and 32%, respectively, greater than control). Nonhepatic glucose uptake increased significantly during delivery of GLP-1 at 5.1 and 10 pmol. kg(-1). min(-1) (25 and 46% greater than control) and tended (P = 0.1) to increase during GLP-1 infusion at 20 pmol. kg(-1). min(-1) (24% greater than control). Intraportal infusion of GLP-1 at high physiological and pharmacological rates increased glucose disposal primarily in nonhepatic tissues.     

PAL31, a novel nuclear protein, expressed in the developing brain

We cloned a cDNA encoding a novel protein (PAL31) predominantly expressed in the fetal rat brain by differential display. PAL31 contains leucine-rich repeat domains, a highly acidic region and a putative nuclear localization signal. PAL31 has 50-70% similarity to SSP29, APRIL, LANP, PHAP I, and PP32. Expression of PAL31 mRNA in the brain was high during the fetal period and decreased after birth. Immunohistochemical studies showed that PAL31 is expressed in the entire embryonic brain, whereas in the adult brain its expression is restricted to the subventricular zone where there are neural progenitor cells. It was also revealed that PAL31 is colocalized with PCNA in the nucleus, indicating that the PAL31 expression is developmentally regulated. Considering the primary structure of PAL31 and its spatiotemporal expression pattern, PAL31 is a novel nuclear protein related to the development of the brain through the proliferation of neuronal cells.     

Rescue of embryonic lethality in hepatocyte growth factor/scatter factor knockout mice

While the targeted disruption of a gene is a powerful tool for investigating the physiological functions of that gene, disruption of a gene essential for embryogenesis leads to embryonic death. Rescue of the defect(s) causing embryonic death should promote survival, thus permitting further evaluation of the roles that the gene plays later in the developmental process. Disruption of the gene for mouse hepatocyte growth factor/scatter factor (HGF/SF) leads to middle-stage embryonic lethality because of a defect in placental development. Here we report that a single injection of HGF/SF at embryonic day 9.5 (E9.5) into the amniotic cavity of HGF/SF(-/-) embryos rescued the placental defect and resulted in the survival of the embryos until term. Histological analysis suggested that HGF/SF is also required at the late stage of development for tissue organogenesis. Thus, injection of a secreted factor can be a useful method to rescue the defects causing embryonic lethality.     

Restoration of effectiveness of beta-lactams on methicillin-resistant Staphylococcus aureus by tellimagrandin I from rose red

We found that extract from petals of Rosa canina L. (rose red) strikingly reduced the minimum inhibitory concentration of beta-lactams in methicillin-resistant Staphylococcus aureus. We isolated two compounds that reduced the minimum inhibitory concentrations of beta-lactams from the extract, tellimagrandin I and rugosin B. Tellimagrandin I was very effective regarding the reduction of the minimum inhibitory concentration, and rugosin B showed some effect. Tellimagrandin I showed a weak bactericidal action when added together with oxacillin. Judging from the fractional inhibitory concentration index, the effect of tellimagrandin I and oxacillin was synergistic. Tellimagrandin I also significantly reduced the minimum inhibitory concentration of tetracycline in some strains of methicillin-resistant Staphylococcus aureus.     

Expression of human cytochromes P450 1A1 and P450 1A2 as fused enzymes with yeast NADPH-cytochrome P450 oxidoreductase in transgenic tobacco plants

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.     

Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double strand breaks

Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.     

Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties

Sphere formation has been utilized as a way to isolate multipotent stem/progenitor cells from various tissues. However, very few studies on bone marrow-derived spheres have been published and assessed their multipotentiality. In this study, multipotent marrow cell populations were isolated using a three-step method. First, after elimination of hematopoietic cells, murine marrow-derived adherent cells were cultured in plastic dishes until small cells gradually appeared and multiplied. Cells were then cultured under non-adherent conditions and formed spheres that were immunopositive for a neural precursor marker, nestin. RT-PCR analysis also revealed that the spheres were positive for nestin in addition to PPARgamma, osf2, SOX9, and myoD, which are markers of precursors of adipocytic, osteoblastic, chondrocytic, and skeletal myeloblastic lineages, respectively. Finally, spheres were dissociated into single cells and expanded in adherent cultures. Under appropriate induction conditions, the sphere-derived cells acquired the phenotypic properties in vitro of neurons, skeletal myoblasts, and beating cardiomyocytes, as well as adipocytes, osteoblasts, and chondrocytes. Next, sphere-derived cells were transplanted into murine myocardial infarction models. One month later, they had become engrafted as cardiomyocytes, and cardiac catheterization showed significant functional improvements. Thus, sphere-derived cells represent a new approach to enhance the multi-differentiation potential of murine bone marrow.     

A defect in glucose-induced dissociation of glucokinase from the regulatory protein in Zucker diabetic fatty rats in the early stage of diabetes

Effect of stimulation of glucokinase (GK) export from the nucleus by small amounts of sorbitol on hepatic glucose flux in response to elevated plasma glucose was examined in 6-h fasted Zucker diabetic fatty rats at 10 wk of age. Under basal conditions, plasma glucose, insulin, and glucagon were approximately 8 mM, 2,000 pmol/l, and 60 ng/l, respectively. Endogenous glucose production (EGP) was 44 +/- 4 micromol x kg(-1) x min(-1). When plasma glucose was raised to approximately 17 mM, GK was still predominantly localized with its inhibitory protein in the nucleus. EGP was not suppressed. When sorbitol was infused at 5.6 and 16.7 micromol x kg(-1) x min(-1), along with the increase in plasma glucose, GK was exported to the cytoplasm. EGP (23 +/- 19 and 12 +/- 5 micromol x kg(-1) x min(-1)) was suppressed without a decrease in glucose 6-phosphatase flux (145 +/- 23 and 126 +/- 16 vs. 122 +/- 10 micromol x kg(-1) x min(-1) without sorbitol) but increased in glucose phosphorylation as indicated by increases in glucose recycling (122 +/- 17 and 114 +/- 19 vs. 71 +/- 11 microl x kg(-1) x min(-1)), glucose-6-phosphate content (254 +/- 32 and 260 +/- 35 vs. 188 +/- 20 nmol/g liver), fractional contribution of plasma glucose to uridine 5'-diphosphate-glucose flux (43 +/- 8 and 42 +/- 8 vs. 27 +/- 6%), and glycogen synthesis from plasma glucose (20 +/- 4 and 22 +/- 5 vs. 9 +/- 4 mumol glucose/g liver). The decreased glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake may result from failure of the sugar to activate GK by stimulating the translocation of the enzyme.