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Volume 23, Issue 1
Displaying 1-6 of 6 articles from this issue
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Akihiko Tanimura, Yosuke Tojyo, Yoshito Matsumoto, Toshihiko Yajima1998 Volume 23 Issue 1 Pages 1-7
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSWe described methods for imaging the IP3-induced Ca2+ release in Ca2+ storage organelles. IP3-indnced changes in Ca2+ concentrations within Ca2+ stores ([Ca2+]L in permeabilized HSY cells were monitored using the low affinity Ca2+ indicators, mag-fura-2 and mag-fura-red. The ratio images of mag-fura-2 were used to estimate the [Ca2+]L, in the store. The apparent [Ca2+]L, was 300-1000 μM at the cell periphery, whereas the [Ca2+]L in the cytoplasm around the nucleus was 70-150 μM. The [Ca2+]L throughout the cytoplasm was reduced by the application of 10 μM IP3 to 30-70 μM, and could be largely recovered after removal of IP3. The structure of IP3-sensitive Ca2+ stores was investigated by confocal microscopy using mag-fura-red. An IP3-induced increase in fluorescence was observed in the ER-like network and reticulum structures of the cytoplasm, and also in the nuclear envelope, suggesting that these organelles serve as IPs-sensitive Ca2+ stores. An analogous localization of the network and tubular elements of the ER was also demonstrated by electron microscopy. These observations suggest that these fluorescence techniques are useful to study the correlation between the distribution and function of Ca2+ stores.View full abstractDownload PDF (4302K) -
Seishi Maeda, Sang-Yoon Nam, Masahiko Fujisawa, Nobuaki Nakamuta, Kenj ...1998 Volume 23 Issue 1 Pages 9-15
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSExpression of cytoplasmic dynein light-intermediate chain mRNA in the rat testis was examined using in situ hybridization. The ribonucleotide probe, referred to the 5' end of open reading frame (6-515 nucleotids) of cytoplasmic dynein light-intermediate chain 53/55 (LIC-2) of the rat brain (Hughes et al., 1995. J. Cell Sci., 108:17-24), was used. All spermatogenic cells were positive. Pachytene spermatocytes in later stages (afterstage VII) were the most intensely positive and round spermatids were also intense. These findings indicated that all spermatogenic cells may store the light-intermediate chain signal, and spermatocytes may produce it during later stages. The reaction in Sertoli cells was constant in intensity during the spermatogenic cycle, indicating that the light-intermediate chain mRNA signal may have no relation to the stage-dependent organdie transport, and that there may be post-translational regulation of the light-intermediate chain. In interstitium, only a few positive cells were observed. Northern blot hybridization demonstrated that one major band (2.0 kb) and two minor bands (4.4 kb and 3.5 kb) were detected in the testis, while one major band (4.4 kb) and one minor band (3.5 kb) were in the brain. This indicated that there are at least 3 isoforms in cytoplasmic dynein light-intermediate chain 53/55.View full abstractDownload PDF (3613K) -
Sebastiano Miscia, Angela di Baldassarre, Rosa Alba Rana, Anna Maria P ...1998 Volume 23 Issue 1 Pages 17-22
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSPI 3-kinase, an enzyme responsible for the phosphorylation of the D3 position of the inositol ring of phosphatidylinositol (PI), is recognized to be involved in the regulation of many cellular processes such as mitogenic signalling, inhibition of apoptosis, intracellular vesicle trafficking/secretion, regulation of actin and integrin functions and regulation of protein kinases induced by tumour necrosis factor, oncoproteins and ultraviolet light. Here we report the subcellular distribution and the phosphorylative pattern of p85 α subunit of PI 3-kinase in Burkitt lymphoma cells exposed to R interferon α treatment. Immunocytochemical analysis of this enzyme, performed by confocal microscopy, revealed an increased expression of this protein at cytoplasmic level after 90 min of interferon a treatment. Western blotting analyses performed on nuclear and cytoplasmic fractions confirmed the overexpression found by confocal microscopy at cytoplasmic level in the 90 min interferon α treated cells still persisting in the 24 hr treated samples. Such an overexpression was paralleled by an increase of tyrosine phosphorylation both at cytoplasmic and nuclear level suggesting that an enhanced requirement for cytoplasmic expression and phosphorylation of PI 3-kinase might be necessary to the cell for regulating some cytoplasmic-nuclear cross talk involved in the control of Burkitt lymphoma cell metabolism following interferon α treatment.View full abstractDownload PDF (1485K) -
Yasunori Miyamoto, Kaori Hara, Yoshiko Matsumoto, Masao Hayashi1998 Volume 23 Issue 1 Pages 23-32
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSVitronectin in a cell-adhesion molecule whose expression is temporally and spatially regulated in vivo, but whose regulatory mechanism of transcription is unknown. In this study, we characterized the mouse vitronectin gene promoter. Luciferase expression vectors cloned the successive 5'- or 3-deletions of the 5'-flanking region upstream of the luciferase gene and were transfected into the human hepatoma cell line HepG2. The assay of luciferase activity in the transfected cells revealed that a 38 base pair (bp)-element (positions + 3 to + 40) displays promoter activity. A consensus sequence consisting of a TATA box and initiator is shown around the transcription initiation site of the mouse vetronectin gene, but the GC box is not shown. Site-directed or deleted mutagenesis against a consensus sequence of TATA box and initiator could not abolish the promoter activity. These results induce that the putative TATA box and initiator are not involved in the promoter activity, and that the Vitronectin promoter lacks the TATA box, initiator and GC box. To characterize trans-acting factors involved in promoter activity, a DNA fragment (position - 74 to +95) was subjected to gel shift assay using nuclear proteins extracted from HepG2 cells. One shifted band was detected by the gel shift assay, suggesting that a nuclear protein binds to the promoter region. Results of the DNase I foot printing assay and gel shift assay demonstrate that the nuclear proteins can bind to the 38 bp-element, which has promoter activity. The nuclear protein is a putative trans-acting factor involved in transcription initiation.View full abstractDownload PDF (2637K) -
Akitsugu Yamamoto, Yoshihiro Tagawa, Tamotsu Yoshimori, Yoshinori Mori ...1998 Volume 23 Issue 1 Pages 33-42
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSWe studied the effects of bafilomycin A1, a potent and specific inhibitor of vacuolar H+ ATPase (V-ATPase), on the process of autophagy in rat hepatoma cell line, H-4-II-E cells. To induce autophagy, cells were transferred from Dulbecco's modified Eagle medium containing 12% fetal calf serum into Hanks' balanced salt solution. When bafilomycin A1 was added to Hanks' balanced salt solution, endogenous protein degradation was strongly inhibited and numerous autophagosomes accumulated in H-4-II-E cells, whereas autolysosomes decreased in number. Acid phosphatase activity was not detected in the autophagosomes which accumulated in the presence of bafilomycin A1, suggesting that fusion between autophagosomes and lysosomes was disturbed by this drug. Inhibition of the fusion was reversible, and the autophagosomes changed into autolysosomes after the removal of the inhibitor. Bafilomycin AI also prevented the appearance of endocytosed HRP in autophagic vacuoles. These results suggested that acidification of the lumenal space of autophagosomes or lysosomes by V-ATPase is important for the fusion between autophagosomes and lysosomes.View full abstractDownload PDF (8105K) -
Kazuyoshi Uehara1998 Volume 23 Issue 1 Pages 43-48
Published: 1998
Released on J-STAGE: March 27, 2006
JOURNAL FREE ACCESSThere seems to exist a relatively low similarity between the amino acid sequences of the testis and milk, β-1, 4-galactosyltransf erase gene products. However, the predicted higher structures of testis and milk β-1, 4-galactosyltransferase proteins revealed a highly significant similarity in the regions required for enzymatic activities. Testis β-1, 4-galactosyltransferase protein contained a WD repeat similar to the motif of the G protein β subunit type I and a Zn-finger motif at the N- and C-terminal portions of the protein, respectively. Phylogenetic analysis of glycosyltransferase proteins revealed that the ancestral gene of testis and milk β-1, 4-galactosyltransferases and β-l, 3-galactosyltransferase evolved into three different destinations at about the same time on an evolutionary scale.View full abstractDownload PDF (1960K)
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