Serum starvation induces abnormal spindle location,RhoA delocalization,and extension of intercellular bridge with the midbody

Serum starvation induces binucleation in HeLa cells, but the effects of serum starvation on mitosis and the significance of binucleation remain unknown. We investigated the effect of serum starvation on mitosis and analyzed the growth of binucleated cells. The frequency of binucleation caused by cytokinesis failure in DMEM without FBS (0% medium) was higher than that in DMEM with FBS (10% medium). In 0% medium, the metaphase spindle location was off-center, and RhoA localization significantly lacked symmetry. The frequency of the extension of intercellular bridge with the midbody in 0% medium was significantly higher than that in 10% medium. Moreover, all mononucleated mitotic cells caused bipolar mitosis and produced only mononucleated daughter cells, but binucleated cells produced various nucleated cells by multipolar mitosis in 0% medium. These results suggest that serum starvation may have various effects on mitosis, and binucleated cells may be related to formation of aneuploidy.     

Processing pathway deduced from the structures of N-glycans in Carica papaya

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.     

6-formylpterin intracellularly generates hydrogen peroxide and restores the impaired bactericidal activity of human neutrophils.

The effects of 6-formylpterin on the impaired bactericidal activity of human neutrophils were examined ex vivo. When neutrophils isolated from fresh blood were incubated with 6-formylpterin, the intracellular production of hydrogen peroxide (H(2)O(2)) occurred. The H(2)O(2) generation by 6-formylpterin in neutrophils occurred in the presence of diphenyleneiodonium (DPI), an inhibitor of NADPH-oxidase. When neutrophils were incubated with DPI, the killing rate of catalase-positive bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), significantly decreased. This impaired bactericidal activity of the DPI-treated neutrophils was a mimic for chronic granulomatous disease (CGD). However, the killing rate of the DPI-treated neutrophils against E. coli and S. aureus significantly increased when 6-formylpterin was administered. Since 6-formylpterin intracellularly generates H(2)O(2) independent from the NADPH-oxidase, it was considered to improve the impaired bactericidal activity of the DPI-treated neutrophils. The use of 6-formylpterin may serve as an option of therapy for CGD.     

Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication

Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft-associated molecules such as glycosylphosphatidylinositol (GPI)-anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP-1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI-anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB-dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.     

Epstein-Barr Virus Infection in Chronically Inflamed Periapical Granulomas

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/μg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.     

Evidence for Cd101 but not Fcgr1 as candidate for type 1 diabetes locus, Idd10

Among polygenes conferring susceptibility to type 1 diabetes in the NOD mouse, Idd10 on distal chromosome 3 has been shown to be important for disease susceptibility. In this study, we investigated the candidacy of Fcgr1 and Cd101 for Idd10, by congenic mapping and candidate gene sequencing. Among seven NOD-related strains studied, the IIS mouse was found to possess a recombinant Idd10 interval with the same sequence at Fcgr1 as the NOD mouse, but a different sequence at Cd101 from that in the NOD mouse with 10 amino acid substitutions. The frequency of type 1 diabetes in NOD mice congenic for IIS Idd10 (NOD.IISIdd10) was significantly reduced as compared to that in the NOD mouse, despite the presence of the identical Fcgr1 sequence. These data indicate that IIS mice possess a resistant allele at Idd10, and suggest that Cd101, but not Fcgr1, is responsible for the Idd10 effect.