Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase,designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen

To date, 10 members of the UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.     

Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.     

Differential regulation of prostacyclin and thromboxane by dexamethasone and celecoxib during oxidative stress in newborn rabbits

To compare the effects of dexamethasone (Dex) and celecoxib (Cel) on F-isoprostane, prostacyclin (PGI2), and thromboxane A2 (TxA2) following hyperoxia, and hyperoxia followed by recovery in room air (RA), newborn rabbits were exposed to hyperoxia (80-100% oxygen) for 4 days, during which they were treated with saline (Sal, i.m.), Dex (i.m.), vehicle (Veh, PO), or Cel (PO, n = 12 per group). Six animals in each group were sacrificed immediately following hyperoxia, and the remainder allowed to recover in RA for 5 days. The control litters were treated simultaneously in RA with all conditions other than atmospheric oxygen being identical. Blood samples were assayed for 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), 6-keto prostaglandin F1alpha (6-ketoPGF1alpha), and TxB2. Dex and Cel decreased 8-epi-PGF2alpha during hyperoxia and the recovery period. Dex increased 6-ketoPGF2alpha following hyperoxia, while similar increments were noted during recovery with Cel. Although TxB2 was decreased only during the recovery period, TxB2/6-ketoPGF1alpha ratio was lower during hyperoxia and recovery in both treated groups. The effect of Cel on 8-epi-PGF2. and TxA2/PGI2 ratio confirm the formation of a COX-derived F2-isoprostane that is possibly linked to TxA2 receptors. Further studies are required to examine whether Cel can be used as a therapeutic alternative to Dex for oxygen-induced injury in the newborn.     

Omega-carboxyl variants of 7-ketocholesteryl esters are ligands for beta(2)-glycoprotein I and mediate antibody-dependent uptake of oxidized LDL by macrophages

beta(2)-Glycoprotein I (beta(2)-GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that beta(2)-GPI specifically binds to oxidized LDL (oxLDL) and that the beta(2)-GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P. Liu, J. Furukawa, K. Kaihara, J. Inagaki, T. Atsumi, N. Sakairi, T. Yasuda, D. R. Voelker, and T. Koike. 2001. A specific ligand for beta(2)-glycoprotein I mediates autoantibody-dependent uptake of oxidized low density lipoprotein by macrophages. J. Lipid Res. 42: 697-709). In the present study, we demonstrate that omega-carboxylated 7-ketocholesteryl esters are critical for beta(2)-GPI binding. A positive ion mass spectrum of a novel ligand, designated oxLig-2, showed fragmented ions at m/z 383 and 441 in the presence of acetone, which share features of oxLig-1 and 7-ketocholesterol. In the negative ion mode, ions at m/z 627, 625, and 243 were observed. oxLig-2 was most likely 7-ketocholesteryl-12-carboxy (keto) dodecanoate. These ligands were recognized by beta(2)-GPI. Liposome binding to macrophages was significantly increased depending on the ligand's concentration, in the presence of beta(2)-GPI and an anti-beta(2)-GPI Ab. Synthesized variant, 7-ketocholesteryl-13-carboxytridecanoate (13-COOH-7KC), also showed a significant interaction with beta(2)-GPI and a similar binding profile with macrophages. Methylation of the carboxyl function diminished all of the specific ligand interactions with beta(2)-GPI. Thus, omega-carboxyl variants of 7-ketocholesteryl esters can mediate anti-beta(2)-GPI Ab-dependent uptake of oxLDL by macrophages, and autoimmune atherogenesis linked to beta(2)-GPI interaction with oxLDL.     

Sustained activation of MEK1-ERK1/2 pathway in membrane skeleton occurs dependently on cell adhesion in megakaryocytic differentiation

A human megakaryoblastic cell line, CMK, was treated with 12-o-tetradecanoylphorbol-13-acetate (TPA) for differentiation-induction. We examined TPA-induced activation of the MEK1-ERK1/2 pathway in the 100,000g Triton X-insoluble fraction of CMK cells as the membrane skeleton and researched the relation of the MEK1-ERK1/2 activation with integrin expression. We found that this activation was divided into two phases: the first activation occurred transiently in the membrane skeleton fraction of the suspended cell status and diminished after 1h; and the second sustained activation was maintained by cell adhesion. TPA-treated CMK cells revealed increased expression of integrins alphaIIb and beta3 only when the cell adhesion persisted, regardless of the difference of culture substratum. Sustained activation of the MEK1-ERK1/2 pathway is generated in the membrane skeleton by continuous cell adhesion and seems to be essential to TPA-induced megakaryocytic differentiation of CMK cells.     

Phylogenetic position of an obligately chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences

Two form ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes from the obligately autotrophic, marine hydrogen oxidizer Hydrogenovibrio marinus were sequenced. The deduced amino acid sequences of both RuBisCOs revealed that they are similar to those of sulfur oxidizers (Thiobacillus) and a purple sulfur bacterium (Chromatium vinosum). According to the 16S rRNA gene sequences, H. marinus is also affiliated with these microorganisms, members of Thiomicrospira being the closest relatives. Sequence similarities of the 16S rRNA genes and of the RuBisCO genes among these γ-Proteobacteria suggest a common autotrophic ancestry. An ancestor of purple sulfur bacteria might be a common root of H. marinus and related sulfur oxidizers. Received: 17 June 1997 / Accepted: 14 November 1997     

Localization of the Q beta replicase recognition site in MDV-1 RNA

Fragments of MDV-1 RNA (a small, naturally occurring template for Q beta replicase) that were missing nucleotides at either their 5' end or their 3' end were still able to form a complex with Q beta replicase. By assaying the binding ability of fragments of different length, it was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA. Fragments missing nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments missing nucleotides at their 3' end were inactive, indicating that the 3'-terminal region of the template is required for the initiation of RNA synthesis. The nucleotide sequences of both the 3' terminus and the central binding region of MDV-1 (+) RNA are almost identical to sequences at the 3' terminus and at an internal region of Q beta (-) RNA.     

Preparation and antitumor activity of O-palmitoyldextran phosphates,O-palmitoyldextrans,and dextran phosphate

Dextran was modified by introduction of palmitoyl and phosphate groups. The derivatives were analyzed by sequential periodate oxidation—sodium borohydride reduction. Only one of these derivatives showed a significant growth-inhibitory effect when administered alone, while a second derivative showed, in combination therapy with an ineffective dose of Mitomycin C, a marked synergistic effect.     

Flocculation of cell walls of brewer's yeast and effects of metal ions,protein-denaturants and enzyme treatments

Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg²⁺-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca²⁺ or other metal ions. Of the alkali metal ions tested, only Na⁺ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca²⁺ or Sn⁴⁺ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn⁴⁺ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr²⁺ and Ba²⁺ were antagonistic to Ca²⁺ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.     

Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides.

A trypsin-type endopeptidase (Kamiya et al., Biochem. Biophys. Res. Commun. 94:855-860, 1980) responsible for the metabolism of rhodotorucine A, the farnesyl undecapeptide mating pheromone secreted by mating type A cells of Rhodosporidium toruloides, was biologically characterized. Metabolic activity was found to be present exclusively on the cell surface of the pheromone target cell. The activity was highly specific to the pheromone, and a biologically inactive analog which has the complete amino acid sequence of rhodotorucine A but lacks the farnesyl residue was not metabolized by intact cells. Pheromone metabolism was inhibited by trypsin substrates such as tosyl-L-arginine methyl ester. The presence of tosyl-L-arginine methyl ester strongly inhibited the sexual differentiation induced by the pheromone at a concentration which did not affect the vegetative growth of R. toruloides. Pheromone-induced sexual differentiation was also strongly inhibited by a metabolizable analog, rhodotorucine A S-oxide, but not by a non-metabolizable one. In mutants defective in early processes of mating, the decrease in the pheromone metabolic activity correlated well with the extent of loss of sensitivity to the pheromone. Both the pheromone metabolism and the capacity for sexual differentiation of a sterile mutant were restored concomitantly with reversion from the sterile to the fertile phenotype. These results suggested that metabolism of the mating pheromone plays an essential role in the process of sexual differentiation in R. toruloides.