A lower volume culture method for obtaining a larger yield of neuron-like cells from mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent a promising cell source for stem cell therapy to replace neurons damaged by neurodegenerative diseases. A system designed for in vitro neuronal differentiation of MSCs is an indispensable technique, which provides MSC-derived functional neurons for cell-replacement therapies and valuable information in pre-clinical research. This study investigated the effects of reducing the volume of neural induction medium on cell viability and neural differentiation of MSCs. When MSCs were differentiated in low volumes of neural induction medium, rather than using the conventional method, the cell density on culture dishes significantly increased. The % cell death, including apoptosis and necrosis, was significantly lower in the lower volume method than in the conventional method. There were no significant differences between the lower volume and conventional methods in the expression levels of the neuronal marker genes. In an analysis of immunostaining for a mature neuronal marker, no significant difference was detected between the media volumes. These findings demonstrate that neuronal induction of MSCs in low volumes of differentiation medium promoted survival during differentiation and resulted in larger numbers of MSC-derived neurons, compared to the conventional method. This novel lower volume method offers both financial and cell-yield advantages over the conventional method.

Phenotypic changes of AADC-only-immunoreactive cells in the alimentary canal of the laboratory shrew, Suncus murinus, induced by systemic administration of monoamine precursors.

In order to elucidate the function of anti-aromatic acid decarboxylase (AADC)-only-positive cells in the alimentary canal, 5-hydroxy-L-tryptophan (5-HTP) or L-3,4-dihydroxyphenylalanine (L-DOPA) was intraperitoneally injected into the laboratory shrew, Suncus murinus, and immunohistochemical studies were conducted on continuous sections of the alimentary canal using specific antisera against tyrosine hydroxylase (TH), AADC, dopamine (DA), and serotonin (5-HT). AADC-only-positive cells localized to the epithelial layer of the alimentary canal from the stomach to the large intestine. These AADC-only-positive cells became DA- and AADC-positive cells after L-DOPA injection, and 5-HT- and AADC-positive cells after 5-HTP injection. These results strongly indicate that the AADC-only-positive cells in the alimentary canal of Suncus murinus are capable of synthesizing DA and 5-HT simultaneously upon administration of L-DOPA and 5-HTP.

Equilibrium shape of (4)He crystal under zero gravity below 200 mK.

Equilibrium crystal shape is the lowest energy crystal shape that is hardly realized in ordinary crystals because of their slow relaxation. (4)He quantum crystals in a superfluid have been expected as unique exceptions that grow extremely fast at very low temperatures. However, on the ground, gravity considerably deforms the crystals and conceals the equilibrium crystal shape, and thus, gravity-free environment is needed to observe the equilibrium shape of (4)He. We report the relaxation processes of macroscopic (4)He crystals in a superfluid below 200 mK under zero gravity using a parabolic flight of a jet plane. When gravity was removed from a gravity-flattened (4)He crystal, the crystal rapidly transformed into a shape with flat surfaces. Although the relaxation processes were highly dependent on the initial condition, the crystals relaxed to a nearly homothetic shape in the end, indicating that they were truly in an equilibrium shape minimizing the interfacial free energy. Thanks to the equilibrium shape, we were able to determine the Wulff's origin and the size of the c-facet together with the vicinal surface profile next to the c-facet. The c-facet size was extremely small in the quantum crystals, and the facet-like flat surfaces were found to be the vicinal surfaces. At the same time, the interfacial free energy of the a-facet and s-facet was also obtained.

Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: possible role in convergent extension.

The Dlg1 gene encodes a member of the MAGUK protein family involved in the polarization of epithelial cells. Null mutant mice for the Dlg1 gene (Dlg1-/- mice) exhibit respiratory failure and cyanosis, and die soon after birth. However, the cause of this neonatal lethality has not been determined. In the present study, we further examined Dlg1-/- mice and found severe defects in the cardiovascular system, including ventricular septal defect, persistent truncus arteriosus, and double outlet right ventricle, which would cause the neonatal lethality. These cardiovascular phenotypes resemble those of mutant mice lacking planar cell polarity (PCP) genes and support a recent notion that DLG1 is involved in the PCP pathway. We assessed the degree of involvement of DLG1 in the development of other organs, as the cochlea, intestine, and skeleton, in which PCP signaling has been suggested to play a role. In the organ of Corti, tissue elongation was inhibited accompanied by disorganized arrangement of the hair cell rows, while the orientation of the stereocilia bundle was normal. In the sternum, cleft sternum, abnormal calcification pattern of cartilage, and disorganization of chondrocytes were observed. Furthermore, shortening of the intestine, sternum, and long bones of the limbs was observed. These phenotypes of Dlg1-/- mice involving cellular disorganization and insufficient tissue elongation strongly suggest a defect in the convergent extension movements in these mice. Thus, our present results provide a possibility that DLG1 is particularly required for convergent extension among PCP signaling-dependent processes.

Targeted deletion of the C-terminus of the mouse adenomatous polyposis coli tumor suppressor results in neurologic phenotypes related to schizophrenia.

BACKGROUND: Loss of adenomatous polyposis coli (APC) gene function results in constitutive activation of the canonical Wnt pathway and represents the main initiating and rate-limiting event in colorectal tumorigenesis. APC is likely to participate in a wide spectrum of biological functions via its different functional domains and is abundantly expressed in the brain as well as in peripheral tissues. However, the neuronal function of APC is poorly understood. To investigate the functional role of Apc in the central nervous system, we analyzed the neurological phenotypes of Apc1638T/1638T mice, which carry a targeted deletion of the 3' terminal third of Apc that does not affect Wnt signaling. RESULTS: A series of behavioral tests revealed a working memory deficit, increased locomotor activity, reduced anxiety-related behavior, and mildly decreased social interaction in Apc1638T/1638T mice. Apc1638T/1638T mice showed abnormal morphology of the dendritic spines and impaired long-term potentiation of synaptic transmission in the hippocampal CA1 region. Moreover, Apc1638T/1638T mice showed abnormal dopamine and serotonin distribution in the brain. Some of these behavioral and neuronal phenotypes are related to symptoms and endophenotypes of schizophrenia. CONCLUSIONS: Our results demonstrate that the C-terminus of the Apc tumor suppressor plays a critical role in cognitive and neuropsychiatric functioning. This finding suggests a potential functional link between the C-terminus of APC and pathologies of the central nervous system.

Identification of DNA-dependent protein kinase catalytic subunit as a novel interaction partner of lymphocyte enhancer factor 1.

Lymphocyte enhancer factor 1 (LEF1), a member of the LEF/T-cell-specific factor (TCF) family of the high mobility group domain transcription factors, acts downstream in canonical Wnt signaling. Aberrant transactivation of LEF1 contributes to the tumorigenesis of colonic neoplasms, sebaceous skin tumors, and lymphoblastic leukemia. LEF1-associated proteins are crucial for regulating its transcriptional activity. In this study, glutathione-S-transferase pull-down assay and mass spectrometry enabled identification of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel interaction partner for LEF1. The interaction between LEF1 and DNA-PKcs was confirmed using in vivo co-immunoprecipitation. Furthermore, double immunofluorescence observations showed that LEF1 and DNA-PKcs colocalized in the nuclei of colon adenocarcinoma cell lines. Identification of the interaction between LEF1 and DNA-PKcs may provide clues for a novel therapy for cancer treatment as well as for understanding LEF1-mediated transcriptional regulation.

Some fine-structural findings on the thyroid gland in Apc1638T/1638T mice that express a C-terminus lacking truncated Apc.

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc(1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc(1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc(1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc(1638T/1638T) and Apc(+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc(1638T/1638T) mice were significantly different whereas the Apc(+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc(1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc(+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc(1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc(+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc(1638T/1638T) mice and suggest suppressed secretory activities of these cells.

4He crystals in superfluid under zero gravity.

The response of 4He crystals to the rapid reduction of gravity down to practically zero in a superfluid was investigated visually, utilizing the parabolic flight of a jet plane. At a high temperature of 1.6 K, the shape of 4He crystals in the bcc phase did not change with a reduction of gravity during a parabolic period of 20 s, due to the low crystallization rate. At lower temperatures, such as 0.63 K, where the crystallization rate is sufficiently high, the shape of 4He crystals in the hcp phase changed significantly, relaxing to a quasiequilibrium shape under zero gravity, where the c facet became enlarged and the a facet emerged on the surface. The crystal did not detach from the sample cell wall at any time because the adhesive force manifested as partial wetting to the wall was sufficiently strong. Some crystals removed from the wall by an acoustic wave pulse were found to float and drift in the superfluid for approximately 4.2 s under zero gravity, although most of them were quickly reattached to the wall.

The C-terminal domain of the adenomatous polyposis coli (Apc) protein is involved in thyroid morphogenesis and function.

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc ( 1638T/1638T ) mice, which express a truncated Apc that lacks the C-terminal domain. Apc ( 1638T/1638T ) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc ( 1638T/1638T ) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc ( 1638T/1638T ) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc ( 1638T/1638T ) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.

Calreticulin-2 is localized in the lumen of the endoplasmic reticulum but is not a Ca2+ -binding protein.

Calreticulin (CRT)-1 is a major Ca(2+)-buffering protein in the lumen of the endoplasmic reticulum. Human and murine CRT-2 was isolated in 2002, but the subcellular localization and function is still unclear. Here, we studied the intracellular localization and function of CRT-2 with hemagglutinin-tagged (HA-) human CRT-2. Western blotting revealed HA-CRT-2 as a single band at 50 kDa. Using immunofluorescence microscopy of cultured fibroblasts and epithelial cells transfected with HA-CRT-2 cDNA, labeling for HA-CRT-2 was seen as a reticular network with a nuclear envelope pattern that colocalized with calnexin and protein disulfide isomerase. Immunoelectron microscopy confirmed that HA-CRT-2 was localized in the lumen of the endoplasmic reticulum. Stains-all staining, a method to detect Ca(2+)-binding proteins, could not stain the immunoprecipitate of HA-CRT-2, although HA-CRT-1 immunoprecipitate was stained blue. These results indicate that the molecular weight of the non-tagged CRT-2 on SDS-PAGE is 49 kDa, and that CRT-2, as well as CRT-1, is localized in the lumen of the endoplasmic reticulum, but that CRT-2 capacity for Ca(2+)-binding may be absent or much lower than that of CRT-1.

Competition between thermal fluctuations and disorder in the crystallization of 4He in aerogel.

The dynamical transition in the crystallization of 4He in aerogel has been investigated by direct visualization and dynamical phase diagrams have been determined. The crystal-superfluid interface in aerogel advances via creep at high temperatures and avalanches at low temperatures. The transition temperature is higher at a higher interface velocity and lower in higher porosity aerogels. The transition is due to competition between thermal fluctuations and disorder for the crystallization process.

Faraday instability of superfluid surface.

We observed that Faraday waves are parametrically generated on a free surface of superfluid 4He when a sample cell is vibrated vertically. Standing-wave patterns appear on the surface, and their frequencies are one-half the driving frequency. We observed clear threshold amplitudes of the vibration for the instability. The difference in the threshold between the superfluid and the normal fluid is explained by a wall damping.

Loss of ICAT gene function leads to arrest of ureteric bud branching and renal agenesis.

ICAT, inhibitor of beta-catenin and T cell factor, or Ctnnbip1, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT-/-) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT+/+) mouse. In the E12.5-ICAT-/- metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT+/+ metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis.

Requirement of the tumour suppressor APC for the clustering of PSD-95 and AMPA receptors in hippocampal neurons.

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC-PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors; however, the clustering of an N-methyl-D-aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses.

gamma-Tubulin-like molecules in the mouse duodenal epithelium.

A mouse monoclonal antibody (G9, Horio et al. in Cell Motil Cytoskel 44:284-295, 1999) that was raised against the gamma-tubulin from a fission yeast, Schizosaccharomyces pombe, showed a unique staining in the mouse small intestine. Similar to another anti-gamma-tubulin antibody that is commercially available, G9 showed typical dot-like staining corresponding to the microtubule-organizing center in the free cells of the epithelium and the connective tissue under it. In addition, G9 stained the cell-cell contacts in the epithelium. This stained region was not bicellular but tricellular junctions of the enterocytes. This staining was unique to G9 and was diminished on the sample of the mouse small intestine, which had lost most of its filamentous microtubules through the preparation process. The tricellular junction is thought to be the weakest point of the epithelial barrier, and no other junctional structures have been identified except for the central sealing elements extending from the tight junctions between the two cells. Our results suggest the existence of a new molecule underlying the tricellular junctions, which may relate to gamma-tubulin and the microtubules.

[Caveolar endocytosis and virus entry].

The endocytic function of caveolae has been controversial for a long time. However, a real-time-imaging analysis of Simian virus 40 (SV40) 's entry in cells has indicated the existence of caveolar endocytosis during virus entry. The caveolae engulfed SV40 virions begin budding from plasma membrane depending on dynamin. SV40 enclosed in caveolae vesicles move to the caveosome, then to the endoplasmic reticulum. In addition, it was demonstrated that human coronavirus-229E enters the cell through caveolae. This review examines the involvement of caveolae in endocytosis used by the viral entry system.

Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae.

CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37 degrees C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37 degrees C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37 degrees C. The depletion of plasmalemmal cholesterol with methyl beta-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.

Lithium inhibits apoptosis of mouse neural progenitor cells.

As undifferentiated precursor cells in the CNS, neural progenitor cells (NPCs) supply new neurons and glial cells to repair damage within the adult brain. Recently, NPCs were found to undergo apoptosis. In serum-free basic fibroblast growth factor-containing culture medium, primary culture cells from fetal mouse neuroepithelium expressed nestin, and thus might be regarded as NPCs. These NPCs demonstrated apoptosis under electron microscopy and TUNEL assay. Treatment of NPCs with lithium, a specific inhibitor of glycogen synthase kinase 3beta (GSK3beta), significantly suppressed apoptosis. Activity of pro-apoptotic protease caspase-3 was not detected in either lithium-untreated or -treated NPCs. These findings suggest that lithium may protect NPCs against apoptosis by inhibiting caspase-3-independent apoptotic pathways.

The expression of cytokeratin in hepatic stellate cells of the cod.

In the current study, we examined the cytoskeletal architecture of cod hepatic stellate cells. We found that the cod hepatic stellate cells contain abundant cytoplasmic filaments. Deep-etch electron microscopy showed that the major component of the cytoplasmic filaments was intermediate filaments, although microtubules and microfilaments were also found in the cytoplasmic filament bundles. Immunoelectron microscopy revealed the presence of beta-tubulin, alpha-smooth muscle actin, smooth muscle type myosin, desmin and cytokeratin but not vimentin or glial fibrillar acidic protein. These results demonstrate that the cytoplasmic filaments of cod hepatic stellate cells are composed of desmin and cytokeratin intermediate filaments, acto-myosin complexes and microtubules, suggesting that the cod hepatic stellate cells have both contractile and structural functions. The expression of cytokeratin in cod hepatic stellate cells indicates that they serve for mechanical support in the extremely soft liver tissues of cods with their abundant lipids.