A 51 kDa Protein Kinase of Potato Activated with Hyphal Wall Components from Phytophthora infestans

In potato tuber tissue, treatment of fungal elicitor, hyphalwall components (HWC) induces various plant defense reactions.As treatment of protein kinase inhibitor prior to HWC treatmentblocks some of defense reactions induced by HWC, involvementof protein kinases in plant defense induction is proposed. Here,we demonstrate HWC-induced activation of a 51-kDa protein kinase(abbreviated p51-PK) using myelin basic protein as a substratein potato tuber discs. The activity of p51-PK was not detectedin the absence of phosphatase inhibitor, NaF, and p51-PK wasimmuroprecipitated with antibody against phosphotyrosine. Pretreatmentof phospholipase C inhibitor, neomycin, and GTP-binding proteinactivator, mastoparan, partially inhibited the HWC-induced activationof p51-PK, suggesting possible involvement of phospholipaseC and GTP-binding protein in the activation of p51-PK. Exogenouslysupplied elicitors, salicylic acid and arachidonic acid, whichare known to induce various defense responses in potato plants,also activated the protein kinase showing the same migrationas p51-PK on SDS-PAGE and different activation patterns. Theseresults implied that p51-PK might be involved in several signaltransduction pathways leading to plant defense responses initiatedby different stimuli. (Received January 11, 1999; Accepted May 25, 1999)     

Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.     

Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes

Tazarotene-induced protein 3 (TIG3) and HRAS-like suppressor family 2 (HRASLS2) exhibit tumor-suppressing activities and belong to the lecithin retinol acyltransferase (LRAT) protein family. Since Ca²⁺-independent N-acyltransferase and H-rev107 (another tumor suppressor), both of which are members of the LRAT family, have been recently reported to possess catalytic activities related to phospholipid metabolism, we examined possible enzyme activities of human TIG3 and HRASLS2 together with human H-rev107. The purified recombinant proteins of TIG3, HRASLS2, and H-rev107 functioned as phospholipase (PL) A_1/2 in a Ca²⁺-independent manner with maximal activities of 0.53, 0.67, and 2.57 μmol/min/mg of protein, respectively. The proteins were active with various phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), and for most of substrates the PLA₁ activity was much higher than the PLA₂ activity. In addition, HRASLS2 catalyzed N-acylation of PE to form N-acyl-PE and O-acylation of lyso PC to form PC. TIG3 and H-rev107 catalyzed the N-acylation and O-acylation at relatively low rates. Moreover, these three proteins showed different expression profiles in human tissues. These results suggest that the tumor suppressors TIG3, HRASLS2 and H-rev107 are involved in the phospholipid metabolism with different physiological roles.     

Wickerhamomyces edaphicus sp. nov. and Pichia jaroonii sp. nov., two ascomycetous yeast species isolated from forest soil in Thailand

Four yeast strains were isolated from soil in a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima province, Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large-subunit (LSU) rRNA gene, small-subunit rRNA gene and internal transcribed spacer (ITS) region, the three strains (S-29, S-63 and S-80) were found to represent a single species of the genus Wickerhamomyces , which were named Wickerhamomyces edaphicus sp. nov. The type strain is S-29^T (BCC 21231^T=NBRC 101969^T=CBS 10408^T). Strain S-75 represented a novel species of the genus Pichia on the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the LSU rRNA gene, for which the name Pichia jaroonii sp. nov. is proposed. The type strain is S-75^T (BCC 23061^T, NRBC 102180^T=CBS 10930^T).     

Cloning of Lactobacillus plantarum IAM 12477 lysine biosynthetic genes encoding functional aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase

Summary The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase (DHPR), respectively, have been cloned from Lactobacillus plantarum IAM 12477 by heterologous complementation to Escherichia coli mutants. The amino acid sequences of the cloned genes showed considerable similarities to the corresponding protein from other gram-positive bacteria. We identified the amino acids that correspond to key catalytic residues of ASADH, DHPS, and DHPR and found them to be conserved in the protein from L. plantarum. ASADH, DHPS, and DHPR activity was detected in the cell extracts of E. coli mutant harboring each gene, indicating that the cloned genes were functionally expressed in E. coli. The regulation of ASADH, DHPS, and DHPR were studied in the cell extracts of both the E.␣coli mutant harboring the gene and L. plantarum; however, those enzymes were found not to be regulated by the end products of the pathway. This paper represents a portion of the thesis submitted by M. N. Cahyanto to Osaka University as partial fulfillment of the requirements for the PhD degree.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

Preferential inhibition of human phosphodiesterase 4 by ibudilast

Ibudilast ophthalmic solution exhibited an improved clinical efficacy over cromoglycate in the treatment of allergic conjunctivitis. To further characterize its principal mode of action, the phosphodiesterase (PDE) inhibitory profile of ibudilast has been examined using human recombinant enzymes. Ibudilast, but not the other commonly used anti-allergic ophthalmic solutions including cromoglycate, ketotifen, tranilast and levocabastine, potently inhibits purified human PDE4A, 4B, 4C and 4D with IC50 values at 54, 65, 239 and 166 nM, respectively. Ibudilast effectively blocks lipopolysaccharide (LPS)-induced tumor necrosis factor (TNFalpha, IC50 = 6.2 microM) and N-formyl-Met-Leu-Phe (fMLP)-induced leukotriene (LT) B4 biosynthesis (IC50 = 2.5 microM) in human whole blood, which are 3 and 6-fold more potent than cilomilast, respectively. The attenuated inflammatory and allergic responses from the potent and preferential PDE4 inhibition of ibudilast may have contributed significantly to its beneficial pharmacological responses and distinguishes ibudilast from the other ophthalmic solutions in the treatment of ocular allergy.