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A double-stranded RNA (dsRNA) mycovirus was detected in a strain of Alternaria alternata showing impaired growth phenotypes. The A. alternata strain is the Japanese pear pathotype, which produces a host-specific AK-toxin. Sequence analysis of the viral genome dsRNAs revealed that this mycovirus consists of five dsRNAs and is evolutionarily related to members of the family Chrysoviridae; the virus was named Alternaria alternata chrysovirus 1 (AaCV1). AaCV1-ORF2 protein accumulated in dsRNA-high-titer sub-isolates with severely impaired phenotypes; heterologous AaCV1-ORF2 overexpression in Saccharomyces cerevisiae caused growth inhibition. In contrast to this yeast growth inhibition phenomenon, the dsRNA-high-titer isolates displayed enhanced pathogenicity against Japanese pear plants, in accordance with a 13-fold increase in AK-toxin level in one such isolate. These findings indicated that AaCV1 is a novel mycovirus that exhibits two contrasting effects, impairing growth of the host fungus while rendering the host 'hypervirulent' to the plant. more...

Shiga toxin (Stx)–producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non–E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non–E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in en... more...

Strains of the Japanese pear pathotype of Alternaria alternata were screened for double-stranded RNAs (dsRNAs). Four strains had several dsRNAs; strain N18 was associated with several dsRNAs and had impaired growth phenotypes such as irregular mycelium and abnormal pigmentation. We isolated dsRNA-cured isolates from strain N18 by single-conidium isolation. The dsRNA-cured isolates had recovered normal growth and pigmentation. Enlarged vesicles were observed in mycelial cells of the original dsRNA-carrying N18 strain. DAPI nuclear staining revealed regression of the nuclei in dsRNA-carrying N18 cells. These results indicate that the dsRNAs might have negative effects, such as apoptosis-like cell death, on the host fungus. more...

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [KD] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (Ki= 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response toN-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking. more...

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte in- teractions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid mi- crodomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8- treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-per- turbing agents such as methyl--cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 gangli- oside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunoflu- orescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is as- sociated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neu- trophils with IL-8 increased the formation of mi- croaggregates composed of neutrophils and acti- vated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with anti- bodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin. J. Leu- koc. Biol. 81: 1414-1421; 2007. more...

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant. more...

NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets.The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy.The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry.These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets. more...

BackgroundThe intradialytic activation of leukocytes is a major cause of hemodialysis (HD)-associated complications. Contact between blood and HD membranes frequently induces the formation of microaggregates composed of activated platelets and leukocytes, causing leukocyte activation that includes the generation of reactive oxygen species (ROS). This complex formation is mediated primarily by the interaction between P-selectin on activated platelets and its counter-ligands on leukocytes.ObjectiveWe examined the preventive effects of α-tocopherol and glycyrrhizin in vitro against platelet-neutrophil microaggregate formation and neutrophil ROS production induced by HD membranes.Methods and ResultsMicroaggregate formation induced by the incubation of heparinized whole blood with polysulfone (PS) HD membranes was effectively inhibited by α-tocopherol and glycyrrhizin. α-Tocopherol, but not glycyrrhizin, was found to inhibit PS membrane-induced P-selectin expression on the platelet surface; however, glycyrrhizin did inhibit both the formation of neutrophil-platelet microaggregates induced by adenosine diphosphate (ADP) and the adhesion of HL60 leukemic cells to P-selectin-expressing Chinese hamster ovary (CHO) cells, suggesting that glycyrrhizin acts as a competitive inhibitor of P-selectin-mediated cell adhesion. Finally, these compounds almost completely abrogated PS membrane-induced and platelet-dependent ROS production by neutrophils.ConclusionsThese results suggest that α-tocopherol and glycyrrhizin may function as preventive agents of HD-associated leukocyte activation though the modulation of platelet-leukocyte interaction. more...

The formation of platelet-neutrophil microaggregates and successive activation of neutrophils are closely related to hemodialysis-associated complications. The microaggregate is mediated primarily by the interaction between P-selectin (CD62P) expressed on activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1, CD162) expressed on neutrophils. We previously reported that the clustered distribution of PSGL-1 on the cell membranes of chemokine-treated neutrophils caused upregulation of the microaggregate formation. In this study, we found that neutrophils treated with human plasma that had been incubated with hemodialysis membranes greatly enhanced the microaggregate formation. The membrane-treated plasma also induced PSGL-1 to form a cap-like cluster on the neutrophil surface. Analysis of several hemodialysis membranes with different materials indicated that the inducibility for the cap-like cluster formation of PSGL-1 parallels their ability to activate the complement system. Both the enhancement of microaggregate formation and the redistribution of PSGL-1 induced by the hemodialysis membrane-treated plasma were almost completely abrogated in the presence of a specific antagonist for the complement component C5a receptor, W-54011. These results strongly suggest that the generation of anaphylatoxin C5a through complement activation induced by hemodialysis membranes is responsible for the clustered redistribution of PSGL-1 in neutrophils leading to the increase in the platelet-neutrophil microaggregate formation. The present study indicates the importance of synergistic exacerbation of complement activation and platelet-neutrophil microaggregate formation in developing hemodialysis-associated complications. more...