Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

N-linked glycan structures of mouse interferon-beta produced by Bombyx mori larvae

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.     

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.     

Fed-batch fermentation of xylose by a fast-growing mutant of xylose-assimilating recombinant Saccharomyces cerevisiae

Mutants of xylose-assimilating recombinant Saccharomyces cerevisiae carrying the xylose reductase and xylitol dehydrogenase genes on plasmid pEXGD8 were selected, after ethyl methanesulfonate treatment, for their rapid growth on xylose medium. The fastest growing strain (strain IM2) showed a lower activity of xylose reductase but a higher ratio of xylitol dehydrogenase to xylose reductase activities than the parent strain, as well as high xylulokinase activity. Southern hybridization of the chromosomal DNA indicated that plasmid pEXGD8 was integrated into the chromosome of mutant IM2, resulting in an increase in the stability of the cloned genes. In batch fermentation under O₂ limitation, the yield and production rate of ethanol were improved 1.6 and 2.7 times, respectively, compared to the parent strain. In fed-batch culture with slow feeding of xylose and appropriate O₂ supply at a low level, xylitol excreted from the cells was limited and the ethanol yield increased 1.5 times over that in the batch culture, with a high initial concentration of xylose, although the production rate was reduced. The results suggested that slow conversion of xylose to xylitol led to a lower level of intracellular xylitol, resulting in less excretion of xylitol, and an increase in the ethanol yield. It was also observed that the oxidation of xylitol was strongly affected by the O₂ supply.Correspondence to: T. Yoshida     

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.     

Autoantigen-specific CD4+CD28low T cell subset prevents autoimmune exocrinopathy in murine Sjögren's syndrome

Organ-specific autoimmune exocrinopathy resembling Sj?gren's syndrome (SS) that spontaneously develops in NFS/sld mutant mice thymectomized 3 day after birth is dependent on Th1-type CD4+ T cells. We previously reported that a cleavage product of 120-kDa alpha-fodrin may be an important autoantigen in the pathogenesis of SS in both an animal model and the patients. We demonstrate that in an animal model of SS with overt exocrinopathy, a unique CD4+ T cell subset expressing CD28low is dramatically increased in spleen cells before the disease onset, but that the CD4+ T cells of diseased mice were virtually all CD28high. We found that the spleen cells in these mice before the disease onset showed a significant increase in autoantigen-specific T cell proliferation. Analysis of in vitro cytokine production by spleen cells indicated, before the disease onset, severely impaired production of IL-2 and IFN-gamma in the animal model, whereas high levels of IL-4 were observed. Expression of cytokine genes, including IL-4, IL-10, and TGF-beta, was detected in FACS-sorted CD4+CD28low T cells by RT-PCR analysis. Transfer of CD4+CD28low T cells into the animal model actually prevented the development of autoimmune lesions including autoantibody production. These results suggest that a CD4+CD28low T cell subset that is continuously activated by an organ-specific autoantigen may play a regulatory role in the development of organ-specific autoimmune disease in an animal model of SS.     

Candida khmerensis sp. nov., a novel cation-tolerant yeast isolated from dry salted shrimp and sewage in Cambodia

Two cation-tolerant yeasts with powdered colonies, K28-3-2T and K26-1-4, were isolated from dry salted shrimp and sewage, respectively, in Siem Reap province, Cambodia. The D1/D2 sequences of the 26S rDNA data showed that the two isolates were conspecific and related to the Pichia burtonii and Candida fennica. Two isolates were examined by a polyphasic taxonomic approach, including molecular phylogenetic analysis, morphological, physiological and biochemical tests, DNA hybridization and MSP-PCR fingerprinting, in comparison with P. burtonii and C. fennica. The two isolates were found to grow by multilateral budding with true and pseudo-mycelium, to not produce ascospores, and to contain ubiquinone Q-8 similar to that of P. burtonii and C. fennica. The two isolates were not differentiated from the two closest species, P. burtonii and C. fennica, by the phenotypic character examined, except for the cation (Li+)-tolerance. From DNA-DNA reassociation studies, however, the two isolates showed low similarities to the closest two species. Based on D1/D2 sequences of 26S rDNA and DNA-DNA reassociation data, they were shown to be a new distinct species from P. burtonii and C. fennica. Therefore, a novel species is proposed, Candida khmerensis sp. nov., represented by strain K28-3-2T (=JCM 13262(T)=CBS 9784T). The novel species, Candida khmerensis sp. nov. can be clearly distinguished from P. burtonii and C. fennica by either the 26S rDNA D1/D2 or ITS region with 5.8S rDNA sequencing, or by the MSP-PCR fingerprinting pattern.     

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

Mitogenic Effect of the Mannans from Saccharomyces cerevisiae on Mouse Spleen Lymphocytes

The DNA synthetic activities of mannans isolated from two Saccharomyces cerevisiae strains were examined in vitro using spleen cells obtained from normal or nude BALB/c strain mice. A highly branched mannan isolated from the S. cerevisiae wild type strain induced a greater increase in mitogenic activity than those displayed by the mannan of the S. cerevisiae X2180–1A-5 mutant strain which possessed fewer branching moieties. Acid-hydrolyzed wild type strain mannan with two-thirds of the molecular weight of the parent intact mannan showed weak mitogenicity. Increases in the DNA synthetic activities of nude and normal spleen cells were almost the same as that of wild type strain mannan, while nylon wool column-passed spleen cells obtained from both normal and nude mice did not show mitogenicity with this mannan. The results indicated that the mitogenic activity was responsible for the highly branched structure of the wild type strain mannan, and that this mannan is a B-cell mitogen.