Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population

We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2',5'-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS (p=0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position -88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS (p=0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.     

Cytology of pleural effusion associated with disseminated infection caused by varicella-zoster virus in an immunocompromised patient. A case report

BACKGROUND: Pleural effusion caused by varicella-zoster virus (VZV) is rare. We report a case of a woman with acute lymphocytic leukemia (ALL) who developed a pleural effusion caused by VZV infection. CASE: A 55-year-old woman with ALL treated with consolidation therapy developed skin vesicles and a pleural effusion. Pleural fluid smears contained numerous mesothelial cells, which had ground-glass nuclei or eosinophilic nuclear inclusions. Some multinucleated giant cells were also seen. Electron microscopic examination revealed intranuclear virus particles, about 150 nm in diameter, in some mesothelial cells. Tissue samples from the skin, lungs, pleura, liver, pancreas, kidneys and gastrointestinal tract, obtained at autopsy, contained many virus-infected cells. They were positive for VZV glyco-protein 1 by immunohistochemistry. CONCLUSION: VZV infection should be considered in the differential diagnosis of an unexplained exudative pleural effusion, especially in immunocompromised hosts.     

Membrane glycoproteomics of fetal lung fibroblasts using LC/MS

Some aberrant N‐glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N‐glycosylation site were high‐mannose type containing nine mannose residues (M9) and monosialo‐fucosylated biantennary oligosaccharides. Several unexpected N‐glycans, such as fucosylated complex‐type and fucosylated high‐mannose and/or fucosylated pauci‐mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.     

Diverging effects of human recombinant anti-hepatitis C virus (HCV) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/HCV pseudotype

Hepatitis C virus (HCV) is the major causative agent of blood-borne non-A, non-B hepatitis. Although a strong humoral response is detectable within a few weeks of primary infection and during viral persistence, the role played by antibodies against HCV envelope glycoproteins in controlling viral replication is still unclear. We describe how human monoclonal anti-HCV E2 antibody fragments isolated from a chronically HCV-infected patient differ sharply in their abilities to neutralize infection of HepG2 cells by a vesicular stomatitis virus pseudotype bearing HCV envelope glycoproteins. Two clones were able to neutralize the pseudotype virus at a concentration of 10 micro g/ml, while three other clones completely lacked this activity. These data can explain the lack of protection and the possibility of reinfection that occur even in the presence of a strong antiviral antibody response.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Chaperonin GroEL meets the substrate protein as a "load" of the rings

Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.     

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.