Skeletal muscle cells derived from mouse skin cultures.

We have established a novel, simple, and highly reproducible method to generate skeletal muscle cells from mouse skin. Small pieces of skin from the back of mice were cultured in extracellular material-coated dishes in typical culture medium for about 3 weeks. Myotubes formed after about a week, grew into twitching myotubes, and became twitching myotube clumps after 3 weeks. Skeletal muscle cells are formed spontaneously with no induction. Myotubes were immunologically positive for myosin heavy chains, MyoD, and myogenin. Ultrastructural analysis revealed the presence of the sarcomere structure. Furthermore, PAX7+/MyoD- muscle stem cells proliferated around these myotubes, and MyoD+/myogenin+/MHC-- cells were also observed. Moreover, we investigated the formation of skeletal muscle cells from the sialidosis mouse skin, and showed that it is decreased compared to that of the wild type. Our method to generate skeletal muscle cells from skin is thought to be useful for the investigation of muscle cell development and muscle-related disorders.

Liquid chromatography-mass spectrometry with triazole-bonded stationary phase for N-methyl-D-aspartate receptor-related amino acids: development and application in microdialysis studies.

We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as D-Ser, Gly, and L-Glu, which were involved in neurotransmission via the N-methyl-D-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p < 0.0001) between relative standard deviation (%) of the area ratio and the analyte-IS retention time differences. Using the proposed method, we were able to accurately analyze trace amounts of amino acids in MD samples using ISs that were structurally similar to the analytes. Furthermore, we observed that the peripheral administration of S-methyl-L-cysteine, which was an inhibitor of the amino acid transporter Asc-1, caused some amino acid level changes in the rat brain. The proposed LC-MS/MS method can be applied in vivo to study the effects of novel therapeutic agents with monitoring the levels of amino acid neuromodulators, such as Glu, Gly, GABA, and D-Ser, in the brain. Graphical abstract LC-MS/MS analysis of amino acid enantiomers in microdialysis samples from rat striatum using triazole-bonded stationary phase.

Alteration in plasma and striatal levels of d-serine after d-serine administration with or without nicergoline: An in vivo microdialysis study.

AIMS: d-Serine (d-Ser), a co-agonist of N-methyl-d-aspartate receptor (NMDAR), is effective for treating schizophrenia. The present study investigated changes in plasma and striatal d-Ser levels in Sprague-Dawley (SD) rats after intraperitoneal d-Ser administration alone or together with nicergoline (Nic), a commercial cerebral ameliorating drug, using in vivo microdialysis (MD) to explore the function of Nic. MAIN METHODS: Phosphate-buffered saline (PBS) or Nic (0, 1.0, or 3.0 mg/kg) followed by d-Ser (5.0, 10.0, 20.0, and 50.0 mg/kg for PBS or 20.0 mg/kg for Nic) was administered intraperitoneally to male SD rats, and the profiles of d-Ser levels in plasma and striatal MD samples were examined by high-performance liquid chromatography (HPLC) with fluorescence detection. The area under the curve (AUC) for the MD and plasma samples was also calculated and statistically compared among groups. KEY FINDINGS: AUC values of d-Ser increased in a d-Ser dose-dependent manner in plasma samples, while a proportional increase in the AUC values of striatal MD samples was only observed in d-Ser doses up to 20 mg/kg. The Nic co-administered group showed a significant increase in the AUC of plasma d-Ser in a Nic dose-dependent manner, but the AUC in striatal d-Ser significantly decreased with increasing Nic doses suggesting that Nic may prevent excess d-Ser from penetrating the central nervous system (CNS). SIGNIFICANCE: Nic may prevent an excessive distribution of exogenous d-Ser, such as that from a dietary origin, into the CNS by suppressing excitatory neurotransmission through NMDAR.

Effect of risperidone on plasma d-serine concentration in rats post-administered with d-serine.

AIMS: Risperidone (Ris) is a second-generation antipsychotic (SGA) used to treat patients with schizophrenia. Additional interventions that increase plasma d-serine (d-Ser) levels could provide improved amelioration of the negative symptoms of schizophrenia. In the present study, we studied whether Ris pretreatment altered the concentration of plasma d-Ser administered intraperitoneally. In addition, the effects of Ris and its main metabolite, 9-hydroxyrisperidone (9-OHRis), on rat d-amino acid oxidase (DAO) activity were examined in vitro. MATERIALS AND METHODS: Ris (0, 0.5, 1.0, or 3.0mg/kg), followed by d-Ser (20mg/kg), were administered intraperitoneally (i.p.) to male Sprague-Dawley rats, and the time-courses of plasma d-Ser, Ris, and 9-OHRis concentrations were examined. Inhibition of DAO activity in rat cerebellar and kidney preparations by Ris and 9-OHRis were measured spectrophotometrically. KEY FINDINGS: Significant increases in plasma d-Ser levels were observed in rats treated with both Ris and d-Ser. This effect occurred in a Ris dose-dependent manner, and the areas under the plasma d-Ser concentration-time curves were similar in rats treated with Ris (1.0mg/kg) and with a commercial DAO inhibitor, 3-methylpyrazole-5-carboxylic acid (1.0mg/kg). Rat plasma analyses showed that 9-OHRis was rapidly produced from Ris; however, high concentrations of Ris and 9-OHRis produced weak DAO inhibition in vitro, suggesting that some other pharmacological effect of Ris and/or 9-OHRis might contribute to its effects on plasma d-Ser levels. SIGNIFICANCE: The combined administration of Ris and d-Ser may increase plasma d-Ser levels, suggesting that this approach could reduce the dose of d-Ser required for these patients.

Inhibitory mechanism of pancreatic amyloid fibril formation: formation of the complex between tea catechins and the fragment of residues 22-27.

Islet amyloid polypeptide (IAPP) is a major component of pancreatic amyloid deposits associated with type 2 diabetes. Polyphenols contained in plant foods have been found to inhibit amyloid fibril formation of proteins and/or peptides. However, the inhibition mechanism is not clear for a variety of systems. Here the inhibition mechanism of green tea polyphenols, catechins, on amyloid fibril formation of the IAPP fragment (IAPP22-27), which is of sufficient length for formation of ß-sheet-containing amyloid fibrils, was investigated by means of kinetic analysis. A quartz crystal microbalance (QCM) determined that the association constants of gallate-type catechins [epicatechin 3-gallate (ECg) and epigallocatechin 3-gallate] for binding to IAPP22-27 immobilized on the gold plate in QCM were 1 order of magnitude larger than those of the free IAPP22-27 peptide, and also those of epicatechin and epigallocatechin. Kinetic analysis using a two-step autocatalytic reaction mechanism revealed that ECg significantly reduced the rate constants of the first nucleation step of amyloid fibril formation, while the rate of autocatalytic growth was less retarded. (1)H nuclear magnetic resonance studies clarified that a IAPP22-27/ECg complex clearly forms as viewed from the (1)H chemical shift changes and line broadening. Our study suggests that tea catechins specifically inhibit the early stages of amyloid fibril formation to form amyloid nuclei by interacting with the unstructured peptide and that this inhibition mechanism is of great therapeutic value because stabilization of the native state could delay the pathogenesis of amyloid diseases and also the toxicity of the small oligomer (protofibril) is reported to be greater than that of the mature fibril.

Chromosomal dynamics at the Shh locus: limb bud-specific differential regulation of competence and active transcription.

The expression of Sonic hedgehog (Shh) in mouse limb buds is regulated by a long-range enhancer 1 Mb upstream of the Shh promoter. We used 3D-FISH and chromosome conformation capture assays to track changes at the Shh locus and found that long-range promoter-enhancer interactions are specific to limb bud tissues competent to express Shh. However, the Shh locus loops out from its chromosome territory only in the posterior limb bud (zone of polarizing activity or ZPA), where Shh expression is active. Notably, while Shh mRNA is detected throughout the ZPA, enhancer-promoter interactions and looping out were only observed in small fractions of ZPA cells. In situ detection of nascent Shh transcripts and unstable EGFP reporters revealed that active Shh transcription is likewise only seen in a small fraction of ZPA cells. These results suggest that chromosome conformation dynamics at the Shh locus allow transient pulses of Shh transcription.

Interaction of tea catechins with lipid bilayers investigated by a quartz-crystal microbalance analysis.

The quartz-crystal microbalance (QCM) technique was applied to investigate the interaction of tea catechins with lipid bilayers. The association constants obtained from the frequency changes of QCM revealed that (-)epicatechin gallate and (-)epigallocatechin gallate interacted with 1,2-dimyristoyl-sn-glycero-3-phosphocholine ca. 1000 times more strongly than (-)epicatechin and (-)epigallocatechin. The results exhibited good correlation with the strength of biological activity.

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster.

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.