HSP27 and MKBP translocate from the cytosolic to myofibril fraction in ischemic rat heart as demonstrated by immunoblotting. Immunohistochemistry analysis showed that ischemia enhances the Z line labeling of HSP27 and MKBP. Two dimensional gel electrophoresis showed that ischemia increases the hyperphosphorylated form of HSP27. These data suggest that HSP27 and MKBP may be involved in the Z line protection against postischemic reperfusion injury.

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Prolyl 4-hydroxylation, the most important post-translational modification in collagen biosynthesis, is catalyzed by prolyl 4-hydroxylase, an endoplasmic reticulum-resident enzyme. HSP47 is a collagenbinding stress protein which also resides in the endoplasmic reticulum (Nagata, K. and Yamada, K.M. (1986) L Biol. Chem., 261, 7531-7536). Both prolyl 4-hydroxylase and HSP47 interact with procollagen α-chains during their folding and/or modification in the endoplasmic reticulum. Recent study has revealed that a simple collagen model peptide, (Pro-Pro-G1y)_n, is recognized by HSP47 as well as by prolyl 4-hydroxylase in vitro (Koide et al., manuscript submitted). In the present study, we investigated the effect of HSP47 on the prolyl 4-hydroxylation of such collagen model peptides. To monitor the enzymatic hydrovlation of the peptides, we developed a non-RI assay system based on reversed-phase HPLC. When HSP47 was added to the reaction mixture, substrate and less-hydroxylated materials accumdated. This effect depended on the peptide-binding activity of HSP47, because a mutant HSP47 without collagen-binding activity did not show any inhibitory effect on prolyl 4-hydroxylation. Knetic analysis and other biochemical analyses suggest that HSP47 retards the enzymatic reaction competing for the substrate peptide.

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Previously we reported an original method of visualizing the shape of yeast nuclei by the expression of green fluorescent protein (GFP)-tagged Xenopus nucleoplasmin in Saccharomyces cerevisiae. To identify components that determine nuclear structure, we searched for mutants exhibiting abnormal nuclear morphology from a collection of temperature-sensitive yeast strains expressing GFP-tagged nucleoplasmin. Four anu mutant strains (anu1-1, 2-1, 3-1 and 4-1; ANU=abnormal nuclear morphology) that exhibited strikingly different nuclear morphologies at the restrictive temperature as compared to the wild-type were isolated. The nuclei of these mutants were irregularly shaped and often consisted of multiple lobes. ANU1, 3 and 4 were found to encode known factors Sec24p, Sec13p and Sec18p, respectively, all of which are involved in the formation or fusion of intracellular membrane vesicles of protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus. On the other hand, ANU2 was not well characterized. Disruption of ANU2 (Δanu2) was not lethal but conferred temperature-sensitivity for growth. Electron microscopic analysis of anu2-1 cells revealed not only the abnormal nuclear morphology but also excessive accumulation of ER membranes. In addition, both anu2-1 and Δanu2 cells were defective in protein transport between the ER and the Golgi, suggesting that Anu2p has an important role in vesicular transport in the early secretory pathway. Here we show that ANU2 encodes a 34 kDa polypeptide, which shares a 20% sequnce identity with the mammalian ε-COP. Our results suggest that Anu2p is the yeast homologue of mammalian ε-COP and the abrupt accumulation of the ER membrane caused by a blockage of the early protein transport pathway leads to alteration of nuclear morphology of the budding yeast cells.

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Extract of Coprinus disseminatus (pers. Fr.) (C. disseminatus) culture broth (EDCB) inhibits proliferation and induces apoptosis in the human cervical carcinoma cells at 5 μg/ml. To determine whether the cell death induced by the EDCB recruits caspases or not, one of the exclusive pathways in cell death, we examined caspase-3 activity in this cell death process. The activity of caspase-3 was remarkably increased when the cell was treated with EDCB, and this activity was nullified by Z-VAD-FMK, a well known caspase-3 inhibitor. From then results, we would expect the EDCB to contain substances with the ability to induce apoptosis in the human cervical carcinoma cells. The extent of the EDCB induced apoptosis is cell line-dependent.

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Monoclonal antibodies (mAbs) were obtained from hybridoma clones established by cell fusion between mouse myelona cells and spleen cells from a mouse immunized against an affinity-purified 40-kDa component of rat 125-kDa glycoprotein (GP125). Two mAbs designated as 3F2 and 6B4 detected a 40-kDa and a 125-kDa band under reducing and nonreducing conditions, respectively, in extracts prepared from rat, mouse and human tumor cells. Association of the 40-kDa protein with CD98 was revealed by sandwich-type enzyme-linked immuosorbent assay. The two mAbs were strongly reactive with various tumor cells and activated lymphocytes, but were only weakly reactive with resting lymphocytes. Confocal microscopy indicated colocalization of CD98 and the 40-kDa protein defined with 3F2 and 6B4 at the cell surface and perinuclear regions. On immunohistochemical analysis of frozen sections of rat tongue, the anti-rat CD98 mAb B3 selectively stained the basal layer and 3F2 stained the upper epithelial part in addition to the basal layer, indicating the existence of CD98-unlinked 40-kDa protein.

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We have suggested in a previous study using 2-nm colloidal gold labeled-testosterone-bovine serum albumin (testosterone-BSA-gold) that 2-nm gold labeled-steroid hormone-BSA conjugates would be a useful tool for analyzing the mechanism of steroid hormone action (39). In this study, we examined whether hydrocortisone-BSA conjugate (hydrocortisone-BSA) showed a similar distribution to radiolabeled hydrocortisone in vivo, by injecting 2-nm colloidal gold labeled-hydrocortisone-BSA (hydrocortisone-BSA-gold) into the rat tail vein. The hydrocortisone-BSA-gold with silver enhancement became visible as silver deposits under electron microscopy in the nuclei of hepatocytes and hepatic stellate cells but not in Kupffer cells in the liver, and in the thymocytes and thymic reticuloepithelial cells in the thymus of a rat killed 2 h postinjection. The percentage of nuclei showing deposits in the non-target cells, the epithelial cells of the seminal vesicle, was similar to the value in the seminal vesicle of a control rat injected with BSA labeled with 2-nm colloidal gold as reported previously. In the hepatocytes and thymocytes of a control rat not injected, the percentages of nuclei showing deposits were similar to those in the rat injected with testosterone-BSA-gold or BSA-gold as reported previously, but lower than those in the rat injected with hydrocortisone-BSA-gold. These results suggest that hydrocortisone-BSA-gold is useful for the morphological study of hydrocortisone target cells, and imply that BSA conjugated with hydrocortisone can enter the target cell nuclei of the rat. The present study further indicates that the fate of gold labeled-steroid hormone-BSA conjugates may be decided at the cell membrane level.

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