When spores of Dictyostelium discoideum matured through normal development were stained with rhodamine-phalloidin, rod-shaped fluorescence was found across the nucleus and in the cytoplasm. Electron micrographs of the rod shape showed that it consisted of long and straight fibers 12nm in diameter. In contrast, a conventional actin rod, which usually is formed under stress conditions, is composed of short filaments 6nm in diameter, and cannot be stained with fluorescent phalloidin. Hence it can be inferred that the structure appeared in Dictyostelium spores under no stress conditions is a novel actin rod.

View full abstract

Calphostin-C is a compound possessing the ability to inhibit protein kinase C (PKC) by oxidative modification in vitro and to enhance the epidermal growth factor (EGF) receptor phosphorylation in vivo in a light-dependent manner. Here, we found that calphostin-C induced c-fos and c-jun mRNA accumulation in the lung adenocarcinoma cell line A549 in a light-dependent manner. Nuclear run-on assay revealed that this mRNA accumulation took place at the transcription level. However, unlike in vitro, calphostin-C did not inhibit cytosolic PKC activity in vivo, and the gene expression induced by calphostin-C was inhibited by another PKC inhibitor, staurosporine. Thus, it was suggested that calphostin-C activates cytosolic PKC-dependent signaling pathway to the induction of "early-response gene" expression in a light-dependent manner.

View full abstract

Microtubule-nucleating ability of the centrosome in the isolated mitotic apparatus was investigated under the condition that the centrosomal fragment was preincubated with guanine nucleotides or its derivatives. GTP and non-hydrolyzable GTP analogues such as GTP-γS or GMP-PNP consistently supported high activity of the centrosome to nucleate microtubules. On the other hand, GDP substantially suppressed the microtubule-nucleating activity of the centrosomes. Evidence has been presented that this is not simply due to the inhibitory effect of GDP on microtubule assembly. This result is consistent with the idea that GTP binding proteins such as 51 kDa protein and γ-tubulin localized to the pericentriolar materials in the centrosome are competent for the nucleation of microtubules when it is saturated with GTP.

View full abstract

We had earlier identified a 60 kDa nuclear lamin protein (lamin_g unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin_g antigen. While all the eight Mabs reacted with lamin_g antigen in an immunoblot analysis, only three Mabs (A₁₁C₇, A₁₁D₄, C₁F₇) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A₁₁C₇ and A₁₁D₄ showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin_g is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin_g as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin_g antibodies. The selective retention of lamin_g in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

View full abstract

To identify a set of mRNAs that increases with the progression of atherosclerosis, differential hybridization screening of a cDNA library, constructed from total RNA in aortae of Watanabe heritable hyperlipidemic (WHHL) rabbits, was carried out. Here we report that one of the positive clones encodes a rabbit homologue of human CD63 antigen. Nucleotide sequence analysis of the cDNA revealed the primary structure of rabbit lysosomal membrane antigen CD63. Analysis of the mRNA by in situ t hybridization revealed hat a significant amount of the mRNA is accumulated in atherosclerotic lesions of WHHL rabbits. Northern blot analysis of total RNA prepared from aortae of WHHL and normal rabbits indicates that expression of the mRNA increased with age in WHHL rabbits, suggesting that expression of the mRNA is correlated with the progression of atherosclerosis.

View full abstract

To investigate the responsiveness to phorbol ester-mediated malignant cell transformation of Balb/c 3T3 variant clones that were morphologically phorbol ester-sensitive and -resistant, we used an in vitro two-stage transformation assay in which cells were treated with 0.1μg/ml of MCA as an initiator and subsequently with 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. The morphologically TPA-sensitive variant, TR5, and the parent cells showed a relatively low sensitivity to TPA-induced cell transformation, where-as the morphologically TPA-resistant variant, TR4 cells, exhibited 50- to 100-fold higher sensitivity to phorbol ester-induced cell transformation than the parent or the TR5cells. We investigated the effects of TPA on protein kinase C activity, 80 kDa PKC substrate phosphorylation, the organization of actin stress fibers, DNA synthesis, and anchorage-independent growth in the three cell clones. They showed similar responses to these biological and biochemical events, indicating that these PKC-mediated events may not be the causes of the differential responsiveness of the variant cells to TPA-induced cell transformation.
We further examined their responsiveness to growth factor-mediated and spontaneous induction of membrane ruffling. When these cells were stimulated by PDGFin their growing phase, membrane ruffling was rapidly induced in the three cells. However, the PDGF-mediated membrane ruffling was completely suppressed in parent and TR5 but not TR4 cells in the con fluent, contact-inhibited (steady-state) growth phase. Similar responses were observed by other growth factors such as insulin, IGF-I, acidic or basic FGF. In addition, when the cells were cultured beyond confluence in the presence of TPA, spontaneous membrane ruffling was induced continuously up to termination of culture in TR4 but not in parent and TR5 cells. These results suggest that the deficiency in cell contact-mediated inhibition of membrane ruffling may be responsible for hypersensitivity of TR4 cells to TPA-induced cell transformation.

View full abstract

Myoblast cells, C2C12, which is an established cell line from satellite cells of skeletal muscle of C3H mouse, start to fuse and form multinucleated cells (myotubes) and begin to express creatine phosphokinase and myosin, when culture medium is changed from the growth mediumto the differentiation medium. Among the 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged by phase-contrast microscopy, but did not affect the induction of creatine phosphokinase activity. The addition of N-acetylglucosamine or N, N', N"-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to the differentiation medium, recovered this apparently suppressive effect of wheat germ agglutinin on the myotube development. Tachypleus tridentatus (Japanese horseshoe crab) lectin that specifically recognizes N-acetylneuraminic acid, one of the sialic acids, showed no effect on the myotube development. It was suggested that wheat germ agglutinin suppressed the process through recognizing N-acetylglucosamine containing sugar. Surprisingly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomic DNA content, which represents the fusion level, decreased after incubation in the differentiation medium, indicating that even when wheat germ agglutinin was present in the medium, cell fusion, which is the initial step of the myotube formation, occurred. Immunostaining with anti-skeletal muscle myosin antiserum confirmed that the myosin expressing cells actually fused and formed multinucleated cells. Their shape, however, was thin compared to that in the absence of wheat germ agglutinin. We propose that the membrane fusion step to form myotubes is composed of two distinct steps in C2C12 ; one fusion step is to form long and thin myotubes from mononucleated cells and the other one is to develop fat myotubes. Wheat germ agglutinin specifically inhibits the latter fusion step.

View full abstract

To understand the dynamics of synaptic vesicles and synapsin I, we have studied the localization of synapsin I and synaptophysin in resting and stimulated nerve endings by ultracryomicrotomy and colloidal gold-immunocytochemistry. First, we characterized microvesicles in resting nerve endings of the rat neurohypophysis, which was chosen as the model of nerve ending in this study. Synaptophysin was localized in microvesicles that were clustered beneath the plasma membrane. Quick-freeze deep-etching electron microscopy showed that short strands cross-linked microvesicles to each other, which highly resembly the structures observed in our studies of the presynaptic nerve terminals of central and peripheral nervous system and in vitro reconstitution of synapsin I and synaptic vesicles. Immunocytochemistry showed that synapsin I was localized to the region of cluster of microvesicles. Second, using this system, we examined localization of synapsin I and synaptophysin in nerve endings after electrical stimulation. Besides release of neurosecretory granules, clusters of microvesicles disappeared and both microvesicles and synaptophysin were scattered over nerve endings. These changes were also confirmed by quick-freeze, freeze-substitution. Immunocytochemistry of the stimulated sample revealed that synapsin I was also scattered. The results show that microvesicles in neurohypophysis have similar characteristics of typical synaptic vesicles and synapsin I has a role as a scaffold to cross-link microvesicles to be clustered in resting nerve endings. This scaffold of synapsin I was disengaged after stimulation to redistribute microvesicles and synapsin I itself, which may be the mechanism of synapsin I to regulate the availability of synaptic vesicles for release.

View full abstract