Mika Takenouchi

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin.

Background. It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. Methods. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-&bgr;-D-glucopyranosyl)-1,2-di-O-acylglycerol (&bgr;-SQDG) that contains a saturated C18 fatty acid, which is designated as &bgr;-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of &bgr;-SQDG(18:0). Results. &bgr;-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. &bgr;-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, &bgr;-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by &bgr;-SQDG(18:0) treatment. Conclusions. &bgr;-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by &bgr;-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

A NEW MEIOTIC ENDONUCLEASE FROM COPRINUS MEIOCYTES

Two different types of Coprinus meiotic nuclease have been previously reported by the authors which are believed to be involved in meiotic chromosome recombination [1,2]. A third meiotic endonuclease was purified from the cap tissues of the basidiocarp of Coprinus cinereus. The enzyme is a 60 kDa molecule composed of a monopolypeptide as revealed by SDS-PAGE and FPLC-Sephacryl S-300 gel filtration. The enzyme belongs to a type of endonuclease which can preferentially digest single-stranded DNA and requires divalent cations as a co-factor, most commonly Mg2+ ions. In the presence of this co-factor, the enzyme converts the supercoiled plasmid DNA (form I) to both the relaxed form (form II) and the linear form (form III). Ca2+ ions can also function as a co-factor, though, in this case, not only is form I plasmid converted to form II, but a few ladder bands between form I and form II are also produced. The Ca2+ ion effect as a cofactor can be prevented with ATP. Immunohistochemical observation shows that the enzyme is distributed in the surface of the gills, which contain the meiotic tissues. These characteristics clearly differ from those of the meiotic nucleases reported previously.

An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence : purification and characterization

Abstract. In our studies on the role of enzymes in plant DNA replication, recombination, and repair, we isolated from cauliflower (Brassica oleracea L. var. botrytis) inflorescences a single-stranded DNA-specific endonuclease that was inhibited by ATP. The endonuclease, designated cauliflower nuclease II, was purified to near homogeneity through six successive column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl sulfate-polyacry amide gel electrophoresis, activity gel, and gel-filtration column chromatography. The enzyme can cleave a linear or a circular single-stranded DNA but cannot cut or nick a double-stranded DNA. The mode of activity of the nuclease is endonucleolytic and non-processive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus far unclear. While ATPγS and GTP can also inhibit the activity, other ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ionic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca2+, which cannot be replaced by Mg2+ or Mn2+. The features of the enzyme and its relation to plant DNA metabolism are discussed.

PROTECTIVE MECHANISM OF ??-SQAG9 LIPOSOME, A SULFONOGLYCOLIPID EXTRACTED FROM SEA URCHIN INTESTINES, AGAINST HEPATIC ISCHEMIA REPERFUSION INJURY

We previously reported that &bgr;-SQAG9 liposome, a sulfonoglycolipid extracted from sea urchin intestines, had a protective effect against hepatic ischemia reperfusion (I/R) injury. In this study, we made a detailed investigation of this protective effect and its mechanism. Rats were pretreated either with &bgr;-SQAG9 liposome (treated group) or with phosphate-buffered saline solution (control group). Thereafter, they were subjected to partial hepatic I/R. The serum levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and histological damage was evaluated with hematoxylin and eosin staining. To investigate the protective mechanism of &bgr;-SQAG9 liposome on I/R injury, the serum levels and the tissue messenger RNA levels of TNF-&agr; and IL-1&bgr; were measured, and polymorphonuclear neutrophil (PMN) infiltration was histologically evaluated by immunohistochemistry. Moreover, to investigate an interaction between &bgr;-SQAG9 liposome and L-selectin on PMNs, flow cytometric analysis and immunofluorescence were performed. &bgr;-SQAG9 liposome reduced the hepatic I/R injury. The pretreatment with &bgr;-SQAG9 liposome reduced the PMN infiltration into the liver parenchyma. On the other hand, there was no apparent difference in the serum levels and the tissue messenger RNA levels of the proinflammatory cytokines between the two groups. Thus, &bgr;-SQAG9 liposome might reduce the hepatic I/R injury by inhibition of the PMN infiltration into the liver parenchyma, which was independent of the regulation of cytokine production. Moreover, we demonstrated that &bgr;-SQAG9 liposome specifically bound to L-selectin on PMN cell surface, which mediated the PMN infiltration. &bgr;-SQAG9 liposome might competitively antagonize L-selectin on PMNs and suppress the subsequent PMN infiltration, resulting in the reduction in I/R injury.