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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.

Membrane-associated RING-CH 8 (MARCH8) is one of 11 members of the MARCH family of RING finger E3 ubiquitin ligases and down-regulates several membrane proteins (e.g. major histocompatibility complex II [MHC-II], CD86, and transferrin receptor). We recently reported that MARCH8 also targets HIV-1 envelope glycoproteins and acts as an antiviral factor. However, it remains unclear whether other family members might have antiviral functions similar to those of MARCH8. Here we show that MARCH1 and MARCH2 are MARCH family members that reduce virion incorporation of envelope glycoproteins. Infectivity assays revealed that MARCH1 and MARCH2 dose-dependently suppress viral infection. Treatment with type I interferon enhanced endogenous expression levels of MARCH1 and MARCH2 in monocyte-derived macrophages. Expression of these proteins in virus-producing cells decreased the efficiency of viral entry and down-regulated HIV-1 envelope glycoproteins from the cell surface, resulting in reduced incorporation of envelope glycoproteins into virions, as observed in MARCH8 expression. With the demonstration that MARCH1 and MARCH2 are antiviral MARCH family members as presented here, these two proteins join a growing list of host factors that inhibit HIV-1 infection.

A number of recent studies have exploited the sizes and functional properties of microdevices and cellular mechanical components to construct bio-microactuators. We previously developed bio-micropumps powered by cardiomyocytes that utilizes glucose in the medium as chemical energy. To fabricate the pump, however, primary neonatal rat cardiomyocytes are indispensable. The operation of harvesting primary cells is inconvenient and ethically not adequate due to the need for animals sacrifice. In contrast, induced pluripotent stem (iPS) cells are obtained from subcutaneous tissue. Their most significant properties are that they proliferate indefinitely and can be differentiated into many kinds of cells, including cardiomyocytes, and also have no ethical issue differently from ES cells. By exploiting these properties of iPS cells, the above issues will be addressed. Based on this concept, we constructed a system for driving fluids as a principal component of a micropump by differentiating iPS cells into spontaneously beating cardiomyocytes. Cellular contractile force was transmitted to fluid in a microchannel by a tent-like thin membrane. The microchip was irradiated with O 2 plasma and coated with gelatin to attach the cells. Embryoid bodies (EBs) of mouse-derived iPS cells were seeded on the microchip and incubated at 37 °C without Leukemia Inhibitory Factor (LIF) to differentiate them into cardiomyocytes. About 2 weeks later, EB beating and periodical oscillation of fluid in a microchannel connected to a diaphragm chamber was observed. The theoretical flow rate assuming the use of ideal check valves ( Q ) was 6.9 nL/min. Our device presents a reasonable alternative to normal cardiomyocytes for preliminary investigations requiring bio-actuating pumps.

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.

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.

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.

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.

Src family kinases are major non-receptor tyrosine kinase in cells and Src-mediated signal transduction involves various cellular functions. In activation process, Src molecules translocate to cell membrane and the subdomains in Src, SH2 and SH3 domains, are exposed. For Src to serve as a kinase, the interaction with its substrates via SH2 and/or SH3 domains is required. Although the activation mechanism of Src has been well-studied, the dynamics of Src at the cell membrane is still unclear. In this study, we examined the role of Src subdomains, especially SH2 and SH3 domains, on the dynamics of Src at the cell membrane. To achieve this, we constructed PAmCherry-tagged wild-type Src (SrcWT), SrcW121A and SrcR178A mutants that decrease the binding of Src to its substrate(s) via SH3 and SH2 domains, respectively, and traced individual Src molecules in the cell membrane with gentle activation of PAmCherry. SrcWT dynamically moved on the cell membrane in the range of 0.27±0.01 μm2/s within a few seconds. The dynamics of SrcR178A mutant was comparable with that of SrcWT, whereas SrcW121A mutant exhibited less mobility (0.16±0.01 μm2/s) at the cell membrane compared with SrcWT. Since both SrcW121A and SrcR178A mutants showed higher phosphorylation level than SrcWT, the result indicates that the less mobility of SrcW121A in the cell membrane seems not to depend upon Src activation status. We further demonstrate that SrcW121A mutant showed ∼30% increase in the Src molecules residence time at focal adhesion compared with SrcWT, which is mediated by slower dissociation from adhesion site. Taken together with enhanced localization of SrcW121A at focal adhesion, our findings show that the SH3 domain of Src molecules governs dynamics of Src at the cell membrane, which may be involved in the rapid signal transduction in cells.

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.

A simple fed-batch system for cultivating genetically engineered yeast generating lactate under the regulation of the PDC1 promoter was established. Traditional strategies that avoid occurrence of Crabtree effect, such as respiratory quotient (RQ) control or ethanol control, are not applicable to the strain because of reduced generation of ethanol and CO(2) by-products. In this system, the feed rate increased when the pH was5.0, and decreased when the pH was5.0. Using this system, cell yields on sucrose increased by approximately 30% compared to that with the conventional RQ control method, due to the early detection of occurrence of Crabtree effect by pH decrease.

Embryonic stem cell (ESC) is pluripotent cell, which is capable of self-renewal and can differentiate to all types of cells. The ESCs collectively differentiate to the right cell in the right place in a group during tissue development. Because the mouse ESC (mESC) can be considered as a simple spontaneously fluctuating individual that does not have any oscillators or circadian clock, it is thought that the collective differentiation in stem cell conceals the mechanism to generate orderliness from assemble of randomness. In order to find the basic role to generate the collective differentiation, we here investigate relationships of heterogeneity of cells and of colonies during the early differentiation stage in mESC by using single cell microscopy. We collected and analyzed total of 100 images of mESC colony containing approximately 30 cells every day for 8 days after differentiation induction. The cellular state could be defined by the fluorescent intensities of the reporters, Venus and mKate2, respectively reporting expressions of Nanog and Oct4, which are core transcription factors for pluripotency maintenance. The heterogeneity in cellular state could be exhibited not only in individual cell but also in colony. We found that the cell tended to resemble its state to the neighboring cells with some kinds of cooperativity, causing the colonial heterogeneity with restricting the cellular fluctuation in a colony. The quantitative analysis of the experimental data lead us the hypothesis that the imbalance of spontaneous fluctuation in a cell and cooperativity between cells realizes a flashing Brownian ratchet, causing the avalanche-like state transition of differentiation state. The present hypothesis was further reinforced by the numerical toy-model with simple rules. The fluctuation and cooperativity play a role for preserving the fractal heterogeneity in multilayer, and the flashing Brownian ratchet is possibly an intrinsic common feature in collective decision making in fluctuating individuals.

The cell environment is very crowded, containing various molecules, proteins and nucleotides. This crowded condition is an indispensable factor for cellular functions of proteins. In the past, the diffusion coefficient of a chemical probe has been used as an evaluation index of the intracellular crowded condition. However, crowding depends not only on the mobility, but also the density of the crowding agents. We have succeeded in making a yellow fluorescent protein (YFP) that senses crowding density via hydrophobicity by inserting into the YFP a glycine and conjugating it to cyan fluorescent protein (CFP), which is insensitive to Forster resonance energy transfer (FRET) probe. This probe has been named GimRET (Glycine inserted mutant FRET probe). GimRET enabled us to visualize the dynamic changes of the intracellular crowding density during cell division. Because GimRET can distinguish the crowding density from the viscosity of the solution, crowding can be evaluated by the GimRET fluorescence intensity ratio and diffusion coefficient, which respectively reflects the density and mobility of the crowding agents. While the diffusion coefficient of GimRET linearly relates to the fluorescence intensity ratio, the slope also depends on the location of the cell, i.e., nucleus or cytoplasm, indicating that the diffusion coefficient alone is insufficient for defining crowding. Here, we propose the simultaneous observation of the GimRET intensity ratio and the diffusion coefficient as a way to evaluate intracellular crowding.

In the past couple of decades, single molecule measurement techniques have tremendously unveiled dynamic behavior and function of individual motor protein molecules. In many living systems, however, multiple molecules work with sort of cooperativity to increase the energy efficiency and the robustness. To understand the physicochemical mechanism of the motor protein systems, the attention is now shifting to upper hierarchy; from individual elements to cooperative motion of multiple protein molecules. We have proposed a novel technique for simultaneous nanometric tracking of dynamic behavior of multiple motor proteins along a one-dimensional rail protein filament. By simple combination of a total internal reflection fluorescence microscope with an imaging spectrometer, multiple motor proteins labeled with quantum dots of different colors can be simultaneously observed with the millisecond time resolution. Even two or more adjacent motor proteins working within nanometric scale, which is much smaller than spatial resolution of optical microscopy, can be separately tracked. Our microscopy system has successfully tracked one-dimensional processive movement of multiple myosins (myosin VI) with a few nanometers precision, and realized observation of how they were chasing each other back and forth on an actin filament. We also applied the microscopy to dynamic observation of two heads of a single myosin (myosin V), and showed the hand-over-hand walking manner, which is consistent with previous works. Furthermore, we extended the technique to two dimensional tracking and applied it to tracking of multiple myosin V on two-dimensional plane. We believe that this would be a useful tool to investigate cooperative movement of motor protein molecules in nanoscale.

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.

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.

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.

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal targetting signal.

Highly resolved excitation spectra for the self-trapped exciton luminescence (σ, π, and E x bands) and for the 1s free exciton luminescence under the two-photon absorption have been studied in RbI at 2 K. These spectra are composed of broad peaks with fine structures: the broad peaks are attributed to the 2p-, 3p- and 4p-exciton transitions, and the fine structures to the energy splitting of the 2p-exciton state by the envelope-hole coupling. The shapes of these excitation spectra are explained by the process of the two-photon absorption with the simultaneous creation of an LO phonon and by the energy relaxation of the p excitons. The former process is treated with third order perturbation theory.

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.

The Japanese language consists of two radically different writing systems: Kanji, based on Chinese characters (morphograms) and Kana, based on characters of syllables (syllabograms). Generally speaking, Kanji characters simultaneously represent for the reader both a phonetic and a semantic value. On the other hand, the individual Kana characters have no semantic value and Kana words must be read on a syllable by syllable basis, each of these translated into sounds, from which words are constructed and semantic content is understood (Soma et al. 1989 Brain112. 1549–1561.). Kana words thus constitute an extremely demanding phonological task, and they cannot usually be identified at first glance. As these two writing systems during reading rely on phonemic processing to different degrees, different cortical areas would be expected to be activated, providing a unique opportunity for further insight into the mechanism of reading. We have used positron emission tomography (PET) measurements of regional cerebral blood flow (rCBF) for mapping the functional anatomy during reading of Kana and Kanji. Analysis of rCBF changes revealed a significant activation of 3.8% of the left supramarginal/angular gyri region during Kana reading compared with Kanji (p < 0.001), supporting a role of this area in phonological recoding during reading of visually presented words. Furthermore, we found a significant activation of both left, 3.2% (p

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.