Activation of α-1 adrenergic receptors modulates the control of left/right sidedness in rat embryos

Presomite stage rat embryos were cultured for 45–49 hr with medium containing various adrenergic agonists and antagonists. -Norepinephrine but not -norepinephrine (several orders of magnitude less potent than the -isomer at α-1 adrenergic receptors) resulted in a dose-dependent increase of situs inversus similar to that found for phenylephrine, an α-1 adrenergic agonist. Prazosin, an α-1 adrenergic antagonist, inhibited phenylephrine-induced situs inversus in a dose-dependent manner. Neither dexmedetomidine, an α-2 adrenergic agonist, nor isoproterenol, a β adrenergic agonist, caused situs inversus. These results provide pharmacological evidence that stimulation of α-1 but not of α-2 and β adrenergic receptors modulates the control of left/right sidedness in rat embryos.     

Plant growth-regulating activities of batatasin III analogues

Nine bibenzyls and 10 stilbenes were synthesized as analoguesof batatasin III, a growth inhibitor isolated from dormant yambulbils, and examined for their plant growth-regulating activities.The bioassays used were the elongation of dark-grown intactrice coleoptiles, auxin-induced elongation of excised oat coleoptiles,and germination of rape and barnyard grass seeds. In the elongationof intact rice coleoptiles, 3,3'-dihydroxy-5-methoxy- (batatasinIII), 3,5-dimethoxy-3'-nitro-, 4'-bromo-3-nitro-, 3-amino-3'-chloro-,3-amino-4'-chloro-bibenzyls and 3-benzyloxy-4'-bromo-5-methoxy-,3-benzyloxy-3',4'-dichloro-5-methoxy-stilbenes were inhibitory,and 4'-bromo-3-nitrostilbene was promotive at a concentrationof 100 mg/liter. The results obtained by the other bioassayswere qualitatively consistent with these findings, although3-amino-4'- chlorobibenzyl and 4'-bromo-3-nitrostilbene werenot tested in all the bioassays. In the seed germination, which was rather tolerant to the testanalogues, batatasin III was inactive but 3-benzyloxy-4'-bromo-5-methoxy-and 3-benzyloxy-3',4'-dichloro- 5-methoxystilbenes were veryactive. Thus, if substituted properly, bibenzyls and stilbenes are activewithout hydroxyl and methoxyl group(s) as the functional group. ³ Present address: The National Institute for EnvironmentalStudies, Yatabc, Ibaraki 300-21, Japan. (Received November 19, 1975; )     

Human erythrocyte glutathione reductase. I. Purification and properties.

1. Glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC. 1.6.4.2) from human erythrocytes was purified 49 000-fold with an overall yield of 15% and a 280/460 nm absorbance ratio of 6.03. The procedure used was the method of Worthington and Rosemeyer modified by addition of heating and recrystallization. 2. It was concluded from the results of purification, electrofocusing and inhibition studies that glutathione reductase is a single enzyme which used both NADPH and NADH as hydrogen donors. 3. Apoenzyme cross-reacts with the antibody to the holoenzyme but has a slightly reduced affinity to the antibody. Apoenzyme can be removed from the hemolysate by heating and centrifugation without loss of holoenzyme. 4. Indirect immunological assay of the specific activity of the erythrocyte glutathione reductase is possible in the enzyme saturated with FAD.     

The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

VEGFR1-Positive Macrophages Facilitate Liver Repair and Sinusoidal Reconstruction after Hepatic Ischemia/Reperfusion Injury

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK^-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK^-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK^-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK^-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK^-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.     

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.