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We have studied the effects of hydrogen peroxide (H2O2) on the differentiation and maintenance of C2C12 myoblasts. The effects of H2O2 were evaluated by cell viability, total protein concentration, the relative amount of muscle-related proteins, sarcomere structure, and active tension generation. Oxidative stress is one of the major causes of myopathy after exercise and thus establishing the method to evaluate the effects on muscle function is essential. The primary function of striated muscle is to generate force, thus, the measurement of active tension is important in assessing the effect of chemicals on muscle. Among the indices we tested, the sarcomere structure was the most sensitive to the H2O2 exposure while the cell viability was less sensitive. The effects of H2O2 on active tension correlated with a decrease in the amount of muscle proteins. In this study, our results showed that the effect of chemicals on muscle should be measured in multiple ways, including active tension generation, for a better understanding of its physiological impact.

Accurate recognition of antigens by specific T cells is crucial for adaptive immunity to work properly. The activation of a T-cell antigen-specific response by an antigen-presenting cell (APC) has not been clearly measured at a single T-cell level. It is also unknown whether the cell-extrinsic environment alters antigen recognition by a T cell. To measure the activation probability of a single T cell by an APC, we performed a single-cell live imaging assay and found that the activation probability changes depending not only on the antigens but also on the interactions of other T cells with the APC. We found that the specific reactivity of single naïve T cells was poor. However, their antigen-specific reactivity increased drastically when attached to an APC interacting with activated T cells. Activation of T cells was suppressed when regulatory T cells interacted with the APC. These findings suggest that although the ability of APCs to activate an antigen-specific naïve T cell is low at a single-cell level, the surrounding environment of APCs improves the specificity of the bulk response.

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper., Summary: Application of high hydrostatic pressure on somatic cells induce changes in gene expression including upregulation in pluripotency genes.

A novel mechanism for Treg-mediated control of immune responses, The immune system in tolerance maintains cell diversity without responding to self-antigens. Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) inhibit T-cell activation through various molecular mechanisms. However, several key questions are still not resolved, including how Tregs control the immune response on the basis of their self-skewed T-cell receptor repertoire and how Tregs avoid impeding relevant immunity against pathogens. Here, we show that Tregs promote the proliferation of conventional T cells in the presence of excessive co-stimulation when murine T cells are stimulated in vitro with allogeneic antigen-presenting cells (APCs). Antigen-specific Tregs increase the number of cells interacting with dendritic cells (DCs) by increasing the number of viable DCs and the expression of adhesion molecules on DCs. Theoretical simulations and mathematical models representing the dynamics of T-APC interaction and T-cell numbers in a lymph node indicate that Tregs reduce the dissociation probability of T cells from APCs and increase the new association. These functions contribute to tolerance by enhancing the interaction of low-affinity T cells with APCs. Supporting the theoretical analyses, we found that reducing the T-cell numbers in mice increases the ratio of specific T cells among CD4+ T cells after immunization and effectively induces autoimmune diabetes in non obese diabetes mice. Thus, as a critical function, antigen-specific Tregs stabilize the immune state, irrespective of it being tolerant or responsive, by augmenting T-APC interaction. We propose a novel regulation model in which stable tolerance with large heterogeneous populations proceeds to a specific immune response through a transient state with few populations.

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha$$\end{document}α(2-3)Gal\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-3)GalNAc\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-4)Gal\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-4)Glc) and its precursor, asialo-GM1 (Gal\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-3)GalNAc\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-4)Gal\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β(1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

In the 2010s, a novel lectin family with β-trefoil folding has been identified in marine mussels from the family Mytilidae (phylum Mollusca). "MytiLec-1," the lectin described in this chapter, was the first member of this family to be isolated and characterized from the Mediterranean mussel Mytilus galloprovincialis, a commercially and ecologically important species, spread in marine coastal areas worldwide. MytiLec-1 bound to the sugar moiety of globotriose (Gb3: Galα1-4Galβ1-4Glc), an α-galactoside, leading to apoptosis of Gb3-expressing Burkitt's lymphoma cells. Although the primary structure of MytiLec-1 was quite unusual, its three-dimensional structure was arranged as a β-trefoil fold, which is the typical architecture of "Ricin B chain (or R)-type" lectins, which are found in a broad range of organisms. To date, MytiLec-1-like lectins have been exclusively found in a few species of the mollusk family Mytilidae (M. galloprovincialis, M. trossulus, M. californianus, and Crenomytilus grayanus) and in the phylum Brachiopoda. Transcriptome data revealed the presence of different structural forms of mytilectin in mussels, which included prototype and chimera-type proteins. The primary sequence of these lectins did not match any previously described known protein family, leading to their assignment to the new "mytilectin family." We here report the method of purification of this lectin and describe its use in cell biology.

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling., Kosuke Watari et al. show that N-myc downstream-regulated gene 1 (NDRG1) stimulates new blood vessel formation that is induced by VEGF-A, using Ndrg1 knockout mice. They find that PLCγ1/ERK signaling mediates this regulation, providing mechanistic insights into how vascular endothelial cells form new vessels.

Machine learning-based cell classifiers use cell images to automate cell-type discrimination, which is increasingly becoming beneficial in biological studies and biomedical applications. Brightfield or fluorescence images are generally employed as the classifier input variables. We propose to use Raman spectral images and a method to extract features from these spatial patterns and explore the value of this information for cell discrimination. Raman images provide information regarding distribution of chemical compounds of the considered biological entity. Since each spectral wavelength can be used to reconstruct the distribution of a given compound, spectral images provide multiple channels of information, each representing a different pattern, in contrast to brightfield and fluorescence images. Using a dataset of single living cells, we demonstrate that the spatial information can be ranked by a Fisher discriminant score, and that the top-ranked features can accurately classify cell types. This method is compared with the conventional Raman spectral analysis. We also propose to combine the information from whole spectral analyses and selected spatial features and show that this yields higher classification accuracy. This method provides the basis for a novel and systematic analysis of cell-type investigation using Raman spectral imaging, which may benefit several studies and biomedical applications.

Type 2 diabetes mellitus (T2DM) is a chronic disease, and dietary modification is a crucial part of disease management. Okara is a sustainable source of fibre-rich food. Most of the valorization research on okara focused more on the physical attributes instead of the possible health attributes. The fermentation of okara using microbes originated from food source, such as tea, sake, sufu and yoghurt, were explored here. The aim of this study is to investigate fermented okara as a functional food ingredient to reduce blood glucose levels. Fermented and non-fermented okara extracts were analyzed using the metabolomic approach with UHPLC-QTof-MSE. Statistical analysis demonstrated that the anthraquinones, emodin and physcion, served as potential markers and differentiated Eurotium cristatum fermented okara (ECO) over other choices of microbes. The in-vitro α-glucosidase activity assays and in-vivo mice studies showed that ECO can reduce postprandial blood glucose levels. A 20% ECO loading crispy snack prototype revealed a good nutrition composition and could serve as a fundamental formulation for future antidiabetes recipe development, strengthening the hypothesis that ECO can be used as a novel food ingredient for diabetic management.

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7β1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.

Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process. Keywords: Embryonic stem cells, Stem cell pluripotency, Nesprin

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

In the stem cell research field, the molecular regulatory network used to define cellular states has been extensively studied, however, the general driving force guiding the collective state dynamics remains to be identified from biophysical aspects. Here we monitored the time-development of the cell-state transition at the single-cell and colony levels, simultaneously, during the early differentiation process in mouse embryonic stem cells. Our quantitative analyses revealed that cellular heterogeneity was a result of spontaneous fluctuation of cellular state and cell-cell cooperativity. We considered that the cell state is like a ball fluctuating on a potential landscape, and found that the cooperativity affects the fluctuation. Importantly, the cooperativity temporarily decreased and increased in the intermediate state of cell differentiation, leading to cell-state transition in unison. This process can be explained using the mathematical equation of flashing-ratchet behaviour, which suggests that a general mechanism is driving the collective decision-making of stem cells.

Autosomal recessive polycystic kidney disease (ARPKD) is an important childhood nephropathy, occurring 1 in 20,000 live births. The major clinical phenotypes are expressed in the kidney with dilatation of the collecting ducts, systemic hypertension, and progressive renal insufficiency, and in the liver with biliary dysgenesis, portal tract fibrosis, and portal hypertension. The systemic hypertension has been attributed to enhanced distal sodium reabsorption in the kidney, the structural defects have been ascribed to altered cellular morphology, and fibrosis to increased TGF-β signaling in the kidney and biliary tract, respectively. The pathogenic mechanisms underlying these abnormalities have not been determined. In the current report, we find that disrupting PKHD1 results in altered sub-cellular localization and function of the C2-WWW-HECT domain E3 family of ligases regulating these processes. We also demonstrate altered activity of RhoA and increased TGF-β signaling and ENaC activity. Linking these phenomena, we found that vesicles containing the PKHD1/Pkhd1 gene product, FPC, also contain the NEDD4 ubiquitin ligase interacting protein, NDFIP2, which interacts with multiple members of the C2-WWW-HECT domain E3 family of ligases. Our results provide a mechanistic explanation for both the cellular effects and in vivo phenotypic abnormalities in mice and humans that result from Pkhd1/PKHD1 mutation.

7 pages; International audience; Scientists are always on the lookout for new modalities of information which could reveal new biological features that are useful for deciphering the complexity of biological systems. Here, we introduce Raman spectroscopy as a prime candidate for ecology and evolution. To encourage the integration of this microscopy technique in the field of ecology and evolution, it is crucial to discuss first how Raman spectroscopy fits within the conceptual, technical and pragmatic considerations of ecology and evolution. In this paper, we show that the spectral information holds reliable indicators of intra- and interspecies variations, which can be related to the environment, selective pressures and fitness. Moreover, we show how the technical and pragmatic aspects of this modality (non-destructive, non-labelling, speed, relative low cost, etc.) enable it to be combined with more conventional methodologies. With this paper, we hope to open new avenues of research and extend the scope of available methodologies used in ecology and evolution.

Our current understanding of molecular biology provides a clear picture of how the genome, transcriptome and proteome regulate each other, but how the chemical environment of the cell plays a role in cellular regulation remains much to be studied. Here we show an imaging method using hybrid fluorescence-Raman microscopy that measures the chemical micro-environment associated with protein expression patterns in a living cell. Simultaneous detection of fluorescence and Raman signals, realised by spectrally separating the two modes through the single photon anti-Stokes fluorescence emission of fluorescent proteins, enables the accurate correlation of the chemical fingerprint of a specimen to its physiological state. Subsequent experiments revealed the slight chemical differences that enabled the chemical profiling of mouse embryonic stem cells with and without Oct4 expression. Furthermore, using the fluorescent probe as localisation guide, we successfully analysed the detailed chemical content of cell nucleus and Golgi body. The technique can be further applied to a wide range of biomedical studies for the better understanding of chemical events during biological processes.

mRNAs for proteins required in elongated spermatids are considered to be transcribed at an early stage and stored in cytoplasm, presumably in chromatoid body (CB), one type of nuage component (a unique structure that appears and disappears during spermatogenesis), because transcription of genes does not occur at late stages. In elongated spermatids, a large amount of tubulin molecules is required to form microtubules of manchette and flagellum. To investigate the possible role of CB in translation of tubulin mRNA, we performed immunofluorescence and immunoelectron microscopic localization studies of α- and β-tubulin in rat spermatogenic cells. β-tubulin was detected in CB, but α-tubulin was not. Other nuage components present in pachytene spermatocytes (ISPG, IMC, SB) were negative for both α- and β-tubulin. Our findings suggest that: (i) β-tubulin in round spermatids is translated within the CB, whereas α-tubulin is not; (ii) αβ-heterodimers are formed outside CB and incorporated into microtubules of manchette and flagellum.

Background Mytilisepta virgata is a marine mussel commonly found along the coasts of Japan. Although this species has been the subject of occasional studies concerning its ecological role, growth and reproduction, it has been so far almost completely neglected from a genetic and molecular point of view. In the present study we present a high quality de novo assembled transcriptome of the Japanese purplish mussel, which represents the first publicly available collection of expressed sequences for this species. Results The assembled transcriptome comprises almost 50,000 contigs, with a N50 statistics of ~1 kilobase and a high estimated completeness based on the rate of BUSCOs identified, standing as one of the most exhaustive sequence resources available for mytiloid bivalves to date. Overall this data, accompanied by gene expression profiles from gills, digestive gland, mantle rim, foot and posterior adductor muscle, presents an accurate snapshot of the great functional specialization of these five tissues in adult mussels. Conclusions We highlight that one of the most striking features of the M. virgata transcriptome is the high abundance and diversification of lectin-like transcripts, which pertain to different gene families and appear to be expressed in particular in the digestive gland and in the gills. Therefore, these two tissues might be selected as preferential targets for the isolation of molecules with interesting carbohydrate-binding properties. In addition, by molecular phylogenomics, we provide solid evidence in support of the classification of M. virgata within the Brachidontinae subfamily. This result is in agreement with the previously proposed hypothesis that the morphological features traditionally used to group Mytilisepta spp. and Septifer spp. within the same clade are inappropriate due to homoplasy. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4012-z) contains supplementary material, which is available to authorized users.

We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 μm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM.

The acquired immune system, mainly composed of T and B lymphocytes, plays a key role in protecting the host from infection. It is important and technically challenging to identify cell types and their activation status in living and intact immune cells, without staining or killing the cells. Using Raman spectroscopy, we succeeded in discriminating between living T cells and B cells, and visualized the activation status of living T cells without labeling. Although the Raman spectra of T cells and B cells were similar, they could be distinguished by discriminant analysis of the principal components. Raman spectra of activated T cells with anti-CD3 and anti-CD28 antibodies largely differed compared to that of naïve T cells, enabling the prediction of T cell activation status at a single cell level. Our analysis revealed that the spectra of individual T cells gradually change from the pattern of naïve T cells to that of activated T cells during the first 24 h of activation, indicating that changes in Raman spectra reflect slow changes rather than rapid changes in cell state during activation. Our results indicate that the Raman spectrum enables the detection of dynamic changes in individual cell state scattered in a heterogeneous population.

Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

We have developed a micropatterning procedure for single myotubes and demonstrated recovery of patterned myotubes without the use of methods that might cause damage to the cells. Since skeletal muscle is a highly ordered tissue mainly composed of myotubes, analysis of single myotubes is one of the promising approaches for studying the various diseases related to skeletal muscle tissues. However, the analysis of single myotubes is quite complicated because of the difficulty in distinguishing individual myotubes differentiated on a normal cell culture surface. In the present study, thin polydimethylsiloxane (PDMS) membranes, which have rectangular holes (30, 50, 100, and 200 microm in width; 500, 750, and 1000 microm in length) through them, were fabricated by using a photolithography technique and used for single myotube micropatterning. A bovine serum albumin-coated (BSA-coated) stencil membrane was placed on a cell culture surface and C2C12 myoblasts were seeded on it. Since the cells could not attach to the surface of the stencil membrane, the cell proliferated and differentiated into myotubes in the hole areas specifically. By peeling off the membrane, a micropattern of myotubes was obtained. It was revealed that the optimum width of rectangular holes for a micropattern of single myotubes was between 30 to 50 microm. Furthermore, by placing a membrane on a thermoresponsive culture surface, recovery of the micropatterned myotubes was possible by lowering the temperature. This method involving the stencil membranes and a thermoresponsive culture surface is useful for analyzing subcellular or single myotubes.

We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events., Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be “static retention” in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.

Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering.

We have fabricated a simple Si-MEMS device consisting of a microcantilever and a base to measure active tension generated by skeletal muscle myotubes derived from murine myoblast cell line C2C12. We have developed a fabrication process for integration of myotubes onto the device. To position myotubes over the gap between the cantilever and the base without damage due to mechanical peeling or the use of an enzymatic reaction, we cultured myotubes on poly-N-isopropylacrylamide (PNIPAAm) as a sacrifice layer. By means of immune staining of alpha-actinin, it was confirmed that a myotube micropatterned onto the device bridged the gap between the cantilever and the base. After 7d differentiation, the myotube was actuated by electrical stimulation. The active tension generated by the myotube was evaluated by measuring the bending of the cantilever using image processing. On twitch stimulation, the myotube on the device contracted and generated active tension in response to the electrical signals. On tetanus tension measurement, approximately 1.0 microN per single myotube was obtained. The device developed here can be used in wide area of in vitro skeletal muscle studies, such as drug screening, physiology, regenerative medicine, etc.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon–intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon–intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes. J. Exp. Zool. (Mol. Dev. Evol.) 312B:95–107, 2009. © 2008 Wiley-Liss, Inc.

The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca(2+) transients. To investigate the relationship between Ca(2+) transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca(2+) transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca(2+) transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca(2+) oscillations, but a sustained increase in intracellular Ca(2+) (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca(2+) oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca(2+) oscillatory signal may be decoded through reorganization of the integrin-cytoskeletal protein complex via calpain-mediated proteolysis.

MytiLec, a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis), shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling, caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Localization of Argonaute2 (AGO2) protein—an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in nuage of rat spermatogenic cells—was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments.

Using Raman spectral imaging, we visualized the cell state transition during differentiation and constructed hypothetical potential landscapes for attractors of cellular states on a state space composed of parameters related to the shape of the Raman spectra. As models of differentiation, we used the myogenic C2C12 cell line and mouse embryonic stem cells. Raman spectral imaging can validate the amounts and locations of multiple cellular components that describe the cell state such as proteins, nucleic acids and lipids; thus, it can report the state of a single cell. Herein, we visualized the cell state transition during differentiation using Raman spectral imaging of cell nuclei in combination with principal component analysis. During differentiation, cell populations with a seemingly homogeneous cell state before differentiation showed heterogeneity at the early stage of differentiation. At later differentiation stages, the cells returned to a homogeneous cell state that was different from the undifferentiated state. Thus, Raman spectral imaging enables us to illustrate the disappearance and reappearance of an attractor in a differentiation landscape, where cells stochastically fluctuate between states at the early stage of differentiation.

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.

Skinned myocardium (or myofibrils) exhibits auto-oscillations of sarcomere length and developed force called SPOC (SPontaneousOscillatoryContraction) under partial activation conditions. In SPOC, each sarcomere repeats the cycle of slow shortening and rapid lengthening, and the lengthening phase propagates sequentially to the adjacent sarcomeres in waves (SPOC wave). In this study, we analyzed the sarcomeric oscillation in SPOC in skinned myocardium of various animal species (rat, rabbit, dog, pig, and cow) with different heart rates. The period of oscillation, the sarcomere shortening velocity, and the velocity of SPOC wave, strongly correlated with the resting heart rate of the animal species. The shortening velocity in particular was proportional to the resting heart rate. We then examined the motile activity of each cardiac myosin by an in vitro motility assay. The sliding velocity of actin filaments, which is an index of the motile activity of myosin, also correlated with the resting heart rate but the relationship was not proportional. As a result, the ratio of sarcomere shortening velocity in SPOC to the sliding velocity of actin filaments was not constant but became higher with a higher heart rate. This suggests that the sarcomere shortening velocity in SPOC is modulated by some additional factors besides the motile activity of myosin, resulting in the proportional relationship between the shortening velocity of the sarcomere and the resting heart rate.

SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins. Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood. We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins. The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p. In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309). Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q). The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes. The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein. The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport. A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae. mVps2 also interacts with SKD1 and is localized to the E235Q compartment. Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1. We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

The authors tested the hypothesis that the oxygen content of brain tissue is negligible by injecting an intracarotid bolus of 15O-labeled tracer into rats. Under the hypothesis, the clearance rates of 15O radioactivity from the brain after injections of both 15O-labeled water (H215O) and 15O-labeled oxyhemoglobin (HbO15O) should be identical. However, the logarithmic slope of the 15O radioactivity curve after HbO15O injection (0.494 ± 0.071 min-1) was steeper than that after H215O injection (0.406 ± 0.038 min−1) ( P15O may dwell in the brain tissue for a finite period of time before it is eventually metabolized or returned to the blood stream unaltered. These findings contradict assumptions made by models currently used to measure cerebral oxygen metabolism.

LGP85 (LIMP II) is a type III transmembrane glycoprotein that is located primarily in the limiting membranes of lysosomes and late endosomes. Despite being the abundant molecule of these compartments, whether LGP85 merely resides as one of the constituents of these membranes or plays a role in the regulation of endosome and lysosome biogenesis remains unclear. To elucidate these questions, we examined the effects of overexpression of LGP85 on the morphology and membrane traffic of the endosomal/lysosomal system. Here we demonstrate that overexpression of LGP85 causes an enlargement of early endosomes and late endosomes/lysosomes. Such a morphological alteration was not observed by overexpression of other lysosomal membrane proteins, LGP107(LAMP-1) or LGP96 (LAMP-2), reflecting a LGP85-specific function. We further demonstrate that overexpression of LGP85 impairs the endocytic membrane traffic out of these enlarged compartments, which may be correlated with or account for the accumulation of cholesterol observed in these compartments. Interestingly, co-transfection of LGP85 and the dominant-negative form of Rab5b (Rab5bS34N) abolished the formation of large vacuoles, suggesting that the GTP-bound active form of Rab5b is involved in the enlargement of endosomal/lysosomal compartments induced by overexpression of LGP85. Thus,these findings provide important new insights into the role of LGP85 in the biogenesis and the maintenance of endosomes/lysosomes. We conclude that LGP85 may participate in reorganizing the endosomal/lysosomal compartments.

Skinned skeletal and cardiac muscle fibres can be activated by MgADP in the presence of MgATP without Ca2+; the isometric tension is developed in a sigmoidal manner with the addition of MgADP under relaxing conditions. The critical concentrations of MgADP for this MgADP-induced contraction are about 7.5 and 2.6 mM for skeletal and cardiac muscle fibres, respectively. To investigate whether muscle regulatory proteins, myosin light chain 2 (LC2) and troponin C (TnC), play a part in the MgADP-induced contraction, these proteins were partly extracted by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), a chelater of divalent cations, and the MgADP-tension relationship was examined in rabbit psoas and bovine cardiac skinned fibres. We found that the sigmoidal MgADP-tension relationship became hyperbolic after a partial extraction of LC2 (about 30 %) and TnC (about 70 %). Reconstitution with LC2 restored the sigmoidal MgADP-tension relationship of control fibres almost fully in both skeletal and cardiac fibres, whereas reconstitution with TnC alone had no effect. Furthermore, cardiac fibres reconstituted with skeletal LC2 exhibited an MgADP-tension relationship intermediate between skeletal and cardiac fibres. The partial extraction of LC2 and TnC resulted in a reduction of the inhibitory effect of inorganic phosphate (P(i)) on the MgADP-activated tension. Reconstitution with LC2 restored the original P(i)-tension relationship, whereas reconstitution with TnC had no effect. In other words, extraction of LC2 apparently increased the affinity of myosin for MgADP but decreased the affinity for P(i). These results demonstrate that LC2 modulates MgADP-induced activation of actomyosin interaction.

Embryonic stem cell (ESC) pluripotency is maintained by core transcription factors (TFs). Although the expression of these TFs is well documented, their expression dynamics is poorly evaluated. Here, we visualized the dynamics of Nanog and Oct3/4 expression in ESC using fluorescent reporters and found that expression of these TFs change dramatically during culture.

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50–60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.

System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states.

The purpose of this study was to determine the relationship between the increase in local cerebral blood flow during neuronal activation (evoked LCBF) and the baseline flow level. We measured the hemodynamics in alpha-chloralose-anesthetized rats using laser-Doppler flowmetry during somatosensory stimulation under the hypocapnic, normocapnic and hypercapnic conditions. The baseline levels of LCBF and red blood cell (RBC) velocity under hypocapnia (PaCO(2)=26.4+/-1.1 mmHg) were, respectively, 10 and 11% lower than those under normocapnia (PaCO(2)=34.2+/-1.4 mmHg) (P0.01). The evoked response magnitude of LCBF and RBC velocity under hypocapnia were, respectively, 22 and 18% lower than those under normocapnia. There was no significant difference in the baseline level and evoked response magnitude of RBC concentration. On the other hand, the baseline levels of LCBF, RBC velocity and RBC concentration under hypercapnia (PaCO(2)=73.4+/-13.3 mmHg) were, respectively, 47, 24 and 14% higher than those under normocapnia (PaCO(2)=34.7+/-2.5 mmHg) (P0.01). The evoked response magnitude of LCBF, RBC velocity and RBC concentration under hypercapnia were, respectively, 96, 82 and 62% greater than those under normocapnia. After normalization with respect to each baseline level, there was no significant difference in normalized evoked response magnitude of LCBF, RBC velocity and RBC concentration, either between hypocapnic and normocapnic conditions or between hypercapnic and normocapnic conditions, indicating that evoked LCBF is proportional to the baseline flow. These results suggest that the amount of evoked LCBF is not determined by the demand for metabolic substrates.

We measured the field potential and local cerebral blood flow (LCBF) using laser-Doppler flowmetry in alpha-chloralose anesthetized rats during activation of the somatosensory cortex by electrical stimulation of the hind paw under independent administration of additional carbon dioxide and oxygen. The aim of this study was to test the hypothesis that the increase in LCBF during activation of the cortex (evoked LCBF) is not directed toward supplying oxygen for oxidative metabolism. Under the hypercapnic condition (PaCO(2) = 74. 9 +/- 14.3 mmHg), the baseline LCBF was about 46.5% higher than that under the normocapnic condition (PaCO(2) = 35.7 +/- 2.1 mmHg) (p < 0. 001), but after normalization for each baseline (divided by the prestimulus level), there was no significant difference in the peak value and the rise time of normalized evoked LCBF. On the other hand, the baseline level of LCBF under the hyperoxic condition (PaO(2) = 479.4 +/- 77.2 mmHg) was about 5.0% lower than that under the normoxic condition (PaO(2) = 105.5 +/- 7.8 mmHg) (p < 0.01), suggesting mild vasoconstriction under the condition of hyperoxia at rest. The peak value of normalized evoked LCBF under the hyperoxic condition was about 6.5% higher than that under the normoxic condition (p < 0.05). In addition, the rise time of evoked LCBF was earlier under the hyperoxic condition (0.37 +/- 0.16 s) than that under the normoxic condition (0.52 +/- 0.12 s) (p < 0.01). The field potential measured during stimulation under hypercapnic and hyperoxic conditions was not significantly different when compared with that under normal gas conditions. These results support our hypothesis and suggest that the excess oxygen is involved in the mechanism underlying the regulation of LCBF.

The aim of this study was to estimate the timing (latency) of the increase in red blood cell (RBC) velocity and RBC concentration, and the magnitude of response in local cerebral blood flow (LCBF) for neuronal activation. We measured LCBF change during activation of the somatosensory cortex by direct microelectrical stimulation. Electrical stimuli of 5, 10 and 50 Hz of 1 ms pulse with 10-15 microA, were given for 5 s. LCBF, RBC velocity and RBC concentration were monitored by laser-Doppler flowmetry (LDF) in alpha-chloralose anesthetized rats (n = 7). LCBF, RBC velocity and RBC concentration increased nearly proportionally to stimulus frequency, i.e. neuronal activity. LCBF rose approximately 0.5 s after the onset of stimulation, and there was no significant time lag of the latencies among LCBF, RBC velocity and RBC concentration at the same stimulus frequency. We interpret these results to mean that the onset of LCBF increase on cortical activation is reflected by a rapid change in arteriole (resistance vessel) dilation and capillary volume. The data also elucidate the linear relationship between LCBF increase and cortical activity.

Lysosomal membrane glycoproteins carry targeting information in cytoplasmic regions. Two distinct targeting motifs in these regions, GY (glycine-tyrosine) and LI (leucine-isoleucine), have been identified and characterized. Accumulating evidence suggests that the adaptor complexes (AP-1, AP-2, and AP-3) recognize this information in cytoplasmic tails of transmembrane proteins. Here we report two different in vitro analyses (affinity beads and surface plasmon resonance) which revealed specific interaction between the cytoplasmic tail of LGP85 and AP-1 but not so with AP-2. We also noted requirement of the LI motif of the LGP85 tail in binding to the AP-1 complex. Our data and others which indicated the binding of AP-3 to the LIMP II (synonym of LGP85) tail suggest that the cytoplasmic tail of LGP85 interacts with AP-1 at the trans-Golgi network (TGN) and AP-3 at late endosomes, respectively. We propose that this sequential interaction between the lysosomal targeting signal and distinct APs along its transport pathway is responsible for the critical sorting of lysosomal membrane proteins and/or the potential proofreading system of mistargeted molecules.

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti–p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca2+-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15°C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.

The purpose of this study was to examine the timing and magnitude of cerebral blood flow (CBF) responses to neuronal activation. We measured the changes in local CBF (LCBF), red blood cell (RBC) velocity and RBC concentration by laser-Doppler flowmetry (LDF) as well as field potential recordings during activation of the somatosensory cortex of the rat in response to electrical stimulation of the hind paw. Electrical stimuli, 0.1 ms pulses of 1-1.5 mA for 5 s, were applied at 0.2, 0.5, 5, 10 and 50 Hz under alpha-chloralose anesthesia. LCBF showed the maximum increase at 5 Hz, and rose approximately 0.5 s after the onset of stimulation regardless of the frequency. The maximum frequency of the field potentials was also obtained at 5 Hz. During activation of the somatosensory cortex, the onset of rise in RBC concentration did not precede that of RBC velocity, and the peak RBC concentration was noted earlier than that of both LCBF and RBC velocity, suggesting that both arteriolar diameter and active changes in the capillary contributed to the LCBF response.

Skinned skeletal and cardiac muscle fibers exhibit spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi), but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307–2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca2+-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

To investigate the mechanisms regulating polarized vesicle delivery to the cell surface in hepatocytes, we have characterized the endogenous plasma membrane (PM)-associated syntaxins. These integral membrane proteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, the PM is divided into two domains, the apical and basolateral. If syntaxins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain expresses one syntaxin isoform. However, we report that rat hepatocytes express three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the syntaxins exhibit distinct, but overlapping patterns of expression among the PM domains. Syntaxin 4 is primarily expressed at the basolateral surface while syntaxins 2 and 3 are enriched at the apical PM. The immunolocalization of syntaxins 2 and 4 in rat hepatocytes and PM sheets revealed similarly complex patterns of PM expression with enhanced apical staining for both. A significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles. We have used quantitative immunoblotting to determine that the syntaxins are relatively abundant PM molecules (11-260 nM) in rat liver, spleen and kidney. Also, we determined that the syntaxin binding protein, Munc-18, is present at concentrations from 1.5-20 nM in the same tissues. Although this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntaxin function, but also for predicting the specific mechanisms that regulate vesicle targeting in hepatocytes and other tissues.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

Substrate elasticity is a potent regulator of the cell state. Soft substrates have been shown to promote the homogeneous self-renewal of mouse embryonic stem cells through the down-regulation of cell-matrix tractions. We therefore investigated whether soft substrates promote the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. After retroviral infection with five factors, Oct3/4, Klf4, Sox2, Lin28 and Nanog, mouse embryonic fibroblasts (MEFs) were cultured on several artificial substrates of varying elasticity and examined for the expression of pluripotency genes. When MEFs were cultured on soft (0.1 kPa) polyacrylamide gels coated with gelatin, the expressions of Nanog and Oct3/4 genes were higher than in cells cultured on rigid plastic dishes (∼10(6) kPa). The same result was obtained at higher elasticity (0.5 kPa) for adult human dermal fibroblasts (HDFa). We also examined whether reprogramming could be enhanced on soft substrates without exogenous gene introduction, finding that cells cultured on soft substrates in the presence of chemicals known to promote cell reprogramming exhibited up-regulated stem cell markers. These results suggest that controlling the substrate stiffness can enhance the initiation of cell reprogramming, which may lead to effective and reproducible iPS cell production.