Biomedical subjects

Mariko Ikeda

Publications and source records attributed to Mariko Ikeda.

7 recordsLinked to original sources

PubMed2006

Coupled transcription-and-translation in Xenopus oocyte and egg extracts.

Coinjection of T7 promoter-driven plasmids and T7 RNA polymerase (T7 RNAP) into Xenopus oocytes results in robust protein synthesis, due to simultaneous gene transcription-and-translation (TnT) in the oocyte cytoplasm [Geib, S., Sandoz, G., Carlier, E., V. Cornet, Cheynet-Sauvion, V., De Waard, M., 2001. A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase. Receptors Channels 7, 331-343; Tokmakov, A.A., Matsumoto, E., Shirouzu, M., Yokoyama, S., 2006. Coupled cytoplasmic transcription-and-translation--a method of choice for heterologous gene experession in Xenopus oocytes. J. Biotechnol. 122, 5-15]. In the present study, we demonstrate that the TnT reaction of protein synthesis can be reconstituted in cell-free extracts of Xenopus oocytes and eggs. Similar to the reaction in oocytes, the effective coupling of bacteriophage T7 RNAP-mediated transcription with the eukaryotic translation machinery takes place in the Xenopus oocyte and egg extracts. However, the kinetics of protein and RNA production in the extracts are quite different from those observed in oocytes. Potent RNA synthesis in the extracts starts immediately after the addition of T7 promoter-driven DNA and T7 RNAP and continues for about 30 min, followed by RNA degradation. The protein product is detectable in the extracts in 15 min after the initiation of the TnT reaction. Efficient protein synthesis in the extracts continues for about 1h. The productivity of this expression system can be boosted by the additions of an RNase inhibitor and an ATP-regeneration system, and by extract dilution. Kinetic analyses suggested that extending the lifetime of the extracts would further increase their productivity.

Animals↗

PubMed2005

Reconstruction of 3D stacked-up structures by rat small hepatocytes on microporous membranes.

The three-dimensional (3D) culture of hepatocytes is essential for the reconstruction of functional hepatic tissues in vitro. In the present experiment, we developed a 3D-culture method in order to reconstruct hepatic cordlike structures by stacking up two-dimensional (2D) tissues composed of rat small hepatocytes (SHs), which are hepatic progenitor cells. Pairs of membranes were prepared and the cells were separately cultured on each membrane. After the SH colonies had developed, one membrane was inverted on top of the other to form an SH bilayer. Thereafter, we investigated whether the stacked cells were organized into differentiated tissues. In the 3D stacked-up structures, bile canaliculi (BC) started to form and gradually developed into anastomosing networks. Transmission electron microscopy revealed that the SHs of the upper and lower layers adhered to one another, and that BC formed between them. Bile canalicular proteins localized on the lumina of the tubular structures. Furthermore, the cells within the structures exhibited mRNA transcription of the hepatic-differentiation markers and maintained a relatively high level of albumin secretion. We conclude that highly differentiated 3D tissues, including functional BC, can be reconstructed by stacking up layers of SHs. This 3D stacked-up culture is useful for the reconstruction of tissue-engineered livers.

Albumins↗

PubMed2005

Coordinated movement of bile canalicular networks reconstructed by rat small hepatocytes.

Hepatocytes in vivo have a potential for liver regeneration, but it has been very difficult to reconstruct hepatic organoids in vitro. Recent studies have shown that small hepatocytes (SHs) can reconstruct hepatic organoids including functional bile canaliculi (BC). In the present study we analyzed the movement of BC formed in the hepatic organoids, focusing on the coordination of contraction and dilation among cells and the mechanism producing the coordination. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Time-lapse images of BC movements were taken and analyzed in cells treated with or without cytochalasin B (CB). Time-lapse images revealed that all BC, regardless of region contracted in a coordinated manner. Actin filaments were observed along the BC even after the BC networks treated with CB dilated markedly. Microinjection of dye was also carried out to investigate the flow thorough BC. Secreted fluorescein from the injected cell flowed along BC, and gap junctional protein connexin 32 was expressed along BC networks, suggesting cell-to-cell communication. Thus, groups of hepatocytes in the hepatic organoids act in a coordinated manner through intercellular communication.

Animals↗

PubMed2005

Effect of fluid force on vascular cell function.

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to fluid frictional force (shear stress) induced by blood flow, and shear stress affects the intracellular calcium ([Ca2+]i), which initiates cellular responses. Here, we studied the effect of long-term exposure of shear stress on [Ca2+]i responses in cultured ECs by using a confocal laser microscope and calcium indicator. At the initiation of shear stress of 20 dyn/cm2 (0 hr), 27% of the cells exhibited [Ca2+]i responses. This percentage gradually decreased with increasing exposure time, reaching about 4% after 24 hr of exposure. These data indicate that long-term shear-stress exposure affects [Ca2+]i responses in cultured ECs. Furthermore, we studied the effect of magnitude of shear stress on macromolecule uptake. For the low shear-stress, the uptake was enhanced, whereas the uptake was inhibited for higher shear-stress.

Analysis of Variance↗

PubMed2004

Manserin, a novel peptide from secretogranin II in the neuroendocrine system.

We have isolated a novel 40 amino acid neuropeptide, designated manserin, from the rat. Manserin is derived from secretogranin II (SgII), a member of granin acidic secretory protein family by proteolytic processing, as previously reported secretoneurin and EM66. Immunohistochemical analysis using anti-manserin antibody revealed that manserin localized in the endocrine cells of the pituitary anterior lobe, but not in the posterior lobe. Interestingly, manserin never co-localized with ACTH in the anterior pituitary, which is in contrast with SgII, suggesting specific immunoreactivity of the antiserum against manserin. Manserin immunostaining was also observed in the neuronal cells of several hypothalamic nuclei and the neurons in the median eminence. These results suggest that manserin exerts a specific role in the neuroendocrine system.

Amino Acid Sequence↗

PubMed2004

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells.

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

Albumins↗

PubMed2004

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model.

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

Animals↗