Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmins (Syts) III, V, VI, and X are classified as a subclass of Syt, based on their sequence similarities and biochemical properties (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273; Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Although they have been suggested to be involved in vesicular trafficking, as in the role of the Syt I isoform in synaptic vesicle exocytosis, their exact functions remain to be clarified, and even their precise subcellular localization is still a matter of controversy. In this study, we established rat pheochromocytoma (PC12) cell lines that stably express Syts III-, V-, VI-, and X-GFP (green fluorescence protein) fusion proteins, respectively, to determine their precise subcellular localizations. Surprisingly, Syts III-, V-, VI-, and X-GFP proteins were found to be targeted to specific organelles: Syt III-GFP to near the plasma membrane, Syt V-GFP to dense-core vesicles, Syt VI-GFP to endoplasmic reticulum-like structures, and Syt X-GFP to vesicles (other than dense-core vesicles) present in cytoplasm. We showed that Syt V-containing vesicles at the neurites of PC12 cells were processed to exocytosis in a Ca2+-dependent manner. Immunohistochemical analysis further showed that endogenous Syt V was also localized on dense-core vesicles in the mouse brain and specifically expressed in glucagon-positive alpha-cells in mouse pancreatic islets, but not in beta- or delta-cells. Based on these results, we propose that Syt V is a dense-core vesicle-specific Syt isoform that controls a specific type of Ca2+-regulated secretion.     

Synthesis and biological activities of fluorinated chalcone derivatives

We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe(3+)-ADP induced NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-2',4'-dimethoxy chalcone (7) was the most effective compound in the in vitro assay using a human cancer cell line panel (HCC panel) consisting of 39 systems.     

Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads

A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.     

Genotyping the mouse severe combined immunodeficiency mutation using the polymerase chain reaction with confronting two-pair primers (PCR-CTPP).

An allele specific polymerase chain reaction with confronting two-pair primers (PCR-CTPP) was developed as an assay for genotyping the mouse Prkdcscid gene mutation (former name scid). The reverse primer (WR) was designed to include the antisense nucleotide (A) specific for the wild type allele at the 3' end with the counterpart forward primer (F) upstream. The other forward primer (MF) was designed to include the sense nucleotide (A) specific for the Prkdcscid mutation at the 3' end with the other counterpart reverse primer (R) downstream. PCR was performed in a single tube with these two pairs of primers. The products specific for each allele extended by F/WR (101 bp) or MF/R (180 bp) were visualized with common PCR products (257 bp) extended by F/R, and three genotypes of mice (Prkdcscid/Prkdcscid, Prkdcscid/+, and +/+) were clearly distinguished.     

Cytological and Reproductive Studies on Japanese Diplazium (Woodsiaceae; Pteridophyta): Apomictic Reproduction in Diplazium with Evergreen Bi- to Tripinnate Leaves

Diplazium , including polymorphic terrestrial species with evergreen bi- to tripinnate leaves. Diplazium hachijoense, D. virescens var. virescens, var. conterminum, var. okinawaense, and two other unnamed varieties, D. kawakamii var. kawakamii, D. dilatatum var. heterolepia, D. taiwanense, D. × kawabatae (=D. dilatatum × taiwanense), D. × takii (=D. hachijoense × virescens var. virescens), and D.× nakamurae (= D. hachijoense × virescens var. conterminum) are apomictic triploids (2n=n=123). Diplazium amamianum and D. esculentum are sexual diploids (2n=82, n=41) and D. subtripinnatum is a sexual tetraploid (2n= 164, n=82). Diplazium dilatatum var. dilatatum includes both sexual diploid and apomictic triploid populations. Cultivated gametophytes of six triploid taxa produced sporophytes apogamously, confirming their apomictic reproduction. All three putative hybrids, D. × kawabatae, D. × takii, and D. × nakamurae, are triploid, apomictic, and fertile taxa, therefore they are not the result of hybridization between known pairs of Japanese Diplazium plants. Received 16 March 1999/ Accepted in revised form 30 September 1999     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.