Biomedical subjects

Tomoyoshi Yoshigaki

Publications and source records attributed to Tomoyoshi Yoshigaki.

6 recordsLinked to original sources

PubMed2006

Difference in distribution of membrane proteins between low- and high-density secretory granules in parotid acinar cells.

Secretory granules (SGs) are considered to be generated as immature granules and to mature by condensation of their contents. In this study, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules was a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins was markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. Immunoprecipitation with anti-syntaxin6 antibody caused coprecipitation of VAMP2 from the medium-density granule fraction without solubilization, but not from Triton X-100-solubilized fraction, while VAMP4 was coprecipitated from both fractions. Therefore, VAMP2 is present on the same granules, but is separated from syntaxin6 and VAMP4, which are expected to be removed from immature granules. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.

Adrenergic beta-Agonists↗

PubMed2005

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules.

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.

Adrenergic beta-Agonists↗

PubMed2005

Phosphodiesterases 1 and 2 regulate cellular cGMP level in rabbit submandibular gland cells.

In rabbit salivary glands, stimulation of muscarinic cholinergic receptors causes production of cGMP through intracellular Ca2+ and nitric oxide. In this study, we investigated a role of cyclic nucleotide phosphodiesterase (PDE) in regulating the cellular cGMP level by using cells dispersed from the submandibular gland. Methacholine, a cholinergic agonist, rapidly elevated the cGMP level. The elevation was greatly enhanced by IBMX, a non-specific inhibitor for most isoforms of the 11 PDEs. The cGMP level was also elevated by MM-IBMX and EHNA, which inhibit the activities of PDE1 and PDE2, respectively. The elevation by the simultaneous application of the two drugs corresponded to 90% of that by IBMX. Therefore, PDE1 and PDE2 are the main PDEs that act to degrade cGMP in methacholine-stimulated cells. The presence of the two PDEs was confirmed by assaying their activities of the cell lysate. In unstimulated cells, the cGMP level was elevated by MM-IBMX and little elevated by EHNA. While the PDE2 activity was thus low, it was estimated that methacholine increases its activity approximately 50-fold. The strong activation can be explained by the elevation of the cGMP level because PDE2 is a cGMP-stimulated PDE. SNAP, a nitric oxide donor, causes production of cGMP without a receptor-operated increase in intracellular Ca2+ concentration. In SNAP-stimulated cells, MM-IBMX elevated the cGMP level higher than in methacholine-stimulated cells although the PDE1 activity is dependent on Ca2+/calmodulin. Besides Ca2+, other factors may regulate the PDE1 activity in living cells.

1-Methyl-3-isobutylxanthine↗

PubMed2003

Why does a cleavage plane develop parallel to the spindle axis in conical sand dollar eggs? A key question for clarifying the mechanism of contractile ring positioning.

Three types of models have been proposed about how the mitotic apparatus determines the position of the cleavage furrow in animal cells. In the first and second types, the contractile ring appears in a cortical region that least and most astral microtubules reach, respectively. The third type is that the spindle midzone positions the contractile ring. In the previous study, a new model was proposed through analyses of cytokinesis in sand dollar and sea urchin eggs. Gradients of the surface density of microtubule plus ends are assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. In the present study, the validity of each model is examined by simulating the furrow formation in conical sand dollar eggs with the mitotic apparatus oriented perpendicular to the cone axis. The new model predicts that unilateral furrows with cleavage planes roughly parallel to the spindle axis appear between the mitotic apparatus and the vertex besides the normally positioned furrow. The predictions are consistent with the observations by Rappaport & Rappaport (1994, Dev. Biol.164, 258-266). The other three types of models do not predict the formation of the ectopic furrows. Furthermore, it is pointed out that only the new model has the ability to explain the geometrical relationship between the mitotic apparatus and the contractile ring under various experimental conditions. These results strongly suggest the real existence of the membrane proteins postulated in the model.

Animals↗

PubMed2003

Theoretical evidence that more microtubules reach the cortex at the pole than at the equator during anaphase in sea urchin eggs.

Astral microtubules are rapidly elongated during anaphase and telophase in sea urchin eggs. The number of microtubules extending to the cell surface was calculated with a computer. For the calculations, microtubules were assumed to radiate from the astral center uniformly over angles. Although microtubules from two asters freely overlapped around the equator, the number per the unit area, i.e. the surface density, was larger in the polar region than in the equatorial region. The ratio of the theoretically calculated numbers in the two regions was close to the ratio obtained from the ultrastructural observations by Asnes and Schroeder in 1979. When counted in the longitudinal section including two astral centers, the microtubule number was a little larger in the equatorial region than in the polar region. However, the numbers do not represent the surface density because the two-dimensional section contains only a small portion of all the microtubules spreading in the three-dimensional space. The fluorescence image for tubulin, in most cases, provides the microtubule distribution in the longitudinal section. Therefore, from such an image, we cannot judge whether the surface density of astral microtubules is larger at the pole or at the equator.

Anaphase↗

PubMed2002

The cleavage plane will bend when one aster of the mitotic apparatus stops growing in compressed sea urchin eggs.

Astral microtubules are elongated greatly during anaphase and telophase in sea urchin eggs. The surface density of microtubules reaching the cell surface can be defined at each surface point. Gradients of the surface-density function were assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. An equation was constructed to calculate the movements of the membrane proteins on a curved surface. The equation was applied to eggs compressed between a coverslip and a glass slide by regarding the egg shape as an oblate spheroid. The simulations explained the observations that contractile-ring microfilaments locally appeared and then developed into a complete ring in compressed eggs. When one aster in the mitotic apparatus stopped growing during anaphase, the equation predicted that the zone of contractile-ring microfilaments is displaced toward the inactivated aster, curves in the view from above and tapers off toward the cell edge. The curve gets sharper as eggs are compressed more greatly and as microtubules from the growing aster penetrate more deeply into the opposite hemisphere. The predictions were compared with the observations by Ishii and Shimizu in 1995 and by Hamaguchi in 1998 regarding the furrow formation by the asymmetric mitotic apparatus.

Actin Cytoskeleton↗