Replication‐coupled passive DNA demethylation for the erasure of genome imprints in mice

Genome‐wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs) is a critical first step to creating a totipotent epigenome in the germ line. We show here that, contrary to the prevailing model emphasizing active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner largely consistent with replication‐coupled passive DNA demethylation: PGCs erase imprints during their rapid cycling with little de novo or maintenance DNA methylation potential and no apparent major chromatin alterations. Our findings necessitate the re‐evaluation of and provide novel insights into the mechanism of genome‐wide DNA demethylation in PGCs.     

Innervation zones of the upper and lower limb muscles estimated by using multichannel surface EMG

The distribution of innervation zones was investigated in 3 subjects for 17 muscles and 8 muscle groups in the upper and lower limb, by detecting bi-directional propagation of motor unit action potentials (MUAPs) with the multichannel surface electrode array. Clarification of the distribution of innervation zones depended on the ease in detecting the propagation of MUAPs and the actual scattering of innervation zones, which were closely related with muscle morphology with respect to the arrangements of muscle fibers. In muscles having fibers running parallel to each other, such as the biceps brachii, intrinsic hand muscles, vastus lateralis and medialis, tensor fasciae latae, peronei, soleus, tibialis anterior, and hypothenar muscles in the foot, it was relatively easy to detect the propagating MUAPs, and the innervation zones were distributed in a relatively narrow band around muscle belly. On the other hand, in muscles with a complicated structure including pinnation of muscle fibers, in-series muscle fibers and aponeurotic tissues, such as the deltoid, flexors and extensors in the forearm, rectus femoris, sartorius, hamstrings and gastrocnemius, it was more difficult to detect the propagating MUAPs and to identify the innervation zones, which were widely scattered or distributed in complex configurations. The distribution of the innervation zones clarified in the present study can be used to find the optimal location of electrodes in surface EMG recordings and of stimulus electrodes in the functional and therapeutic electrical stimulations. It may also be useful in motor point biopsy for diagnosis of neuromuscular diseases as well as in the botulinum toxin injection for the treatment of spasticity.     

A case of Graves' disease with false hyperthyrotropinemia who developed silent thyroiditis.

We encountered a patient who developed silent thyroiditis during the course of Graves' disease. The diagnosis of silent thyroiditis was made on the basis of a low thyroidal 131I uptake, no response to the thyrotropin releasing hormone (TRH) test, and subsequent hypothyroidism despite the presence of high titers of thyrotropin (TSH) receptor antibody (TRAb) and thyroid stimulating antibody (TSAb). The patient, in addition, had a discrepancy between serum TSH and thyroid hormone values. This was due to the presence of interfering substances that react to mouse IgG in the sera since serum TSH levels were decreased in a dose dependent manner by the addition of increasing amounts of mouse IgG to the sera. It should therefore be noted that silent thyroiditis can develop in patients with Graves' disease. Furthermore, clinicians should be aware that two-site immunoassay kits that use mouse monoclonal antibodies are subject to interference by some substances, possibly antibodies which react to mouse IgG.     

Quantitative correlation between the concentration of photoreactive phytochrome and light-induced formation of adventitious shoots in horse-radish hairy roots

The hairy roots of horse-radish (Armoracia rusticana Gaert., Mey. et Scherb.) were found to contain spectrophotometrically active phytochrome. The absorption maxima of the red-absorbing and the far red-absorbing forms of phytochrome were the same as those of phytochrome I in etiolated tissues and of purified phytochrome A. Red light dramatically decreased the concentration of phytochrome in the hairy roots. The phytochrome was concentrated at the proximal ends of hairy roots and the concentration of phytochrome at each position in hairy roots was quantitatively correlated with the frequency of the light-induced formation of adventitious shoots at the same position. The level of phytochrome reflected the duration of culture in darkness. The increase in the concentration of phytochrome with time in darkness was positively correlated with the frequency of light-induced formation of adventitious shoots. The involvement of phytochromes A and B in the induction by light of budding of adventitious shoots from horse-radish hairy roots is discussed on the basis of the newly revealed correlations and earlier results.     

Action spectrum for light-induced formation of adventitious shoots in hairy roots of horseradish

An action spectrum was determined for lightinduced formation of adventitious shoots in hairy roots of horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) cultured in vitro. Near ultraviolet (350–400 nm), blue (440–460 nm) and red light (600–680 nm) were most effective in inducing formation of adventitious buds. Farred light (730 nm) inhibited the promotive effect of all three wavelength regions. These results are consistent with induction by phytochrome(s) of adventitious shoots in hairy roots of horseradish.     

Differentiation of restriction sites in ribosomal DNA in the genusApodemus

Southern blot analysis of ribosomal DNA (rDNA) from seven species ofApodemus was carried out in order to examine the genetic relationships between the species. Analysis of heterogeneity in rDNA spacers inA. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, andA. peninsulae, using 13 different restriction enzymes and cloned mouse rDNA probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction sites, sequence divergence among the different major repetypes was estimated. Among the seven species ofApodemus examined, the major repetypes ofA. flavicollis andA. sylvaticus were the most closely related, having only 1.0% sequence divergence. These repetypes and those of the remaining five species differ substantially from one another, with 4.3–8.5% divergence.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

A neuronal transmembrane protein LRFN4 complexes with 14-3-3s and NCK1 to induce morphological change in monocytic cells via Rac1-mediated actin cytoskeleton reorganization

We previously reported that leucine-rich repeat and fibronectin type III domain-containing 4 (LRFN4) functioned in migration and morphological change (i.e. cell elongation) of monocytic cells. Here, we examined a molecular mechanism regulating LRFN4-mediated cell elongation. We found that 14-3-3 and NCK proteins complexed with LRFN4, and they were involved in LRFN4-mediated cell elongation. We also identified the regions of LRFN4 interacting with NCK1 and 14-3-3s. Finally, we demonstrated that a Rac1 small GTPase was involved in LRFN4-mediated cell elongation. These results indicated that LRFN4 complexed with 14-3-3s and NCK1 to mediate elongation in monocytic cells via Rac-1-mediated actin cytoskeleton reorganization.     

Influence of contraction force and speed on muscle fiber conduction velocity during dynamic voluntary exercise.

Before using electromyographic (EMG) variables such as muscle fiber conduction velocity (MFCV) and the mean or median frequency (MDF) of an EMG power spectrum as indicators of muscular fatigue during dynamic exercises, it is necessary to determine the influence of a joint angle, contraction force and contraction speed on the EMG variables. If these factors affect the EMG variables, their influence must be removed or compensated for before discussing fatigue. The vastus lateralis of eight normal healthy male adults was studied. EMG signals during non-fatiguing dynamic knee extension exercises were detected with a three-bar active surface electrode array. EMG variables were calculated from the detected signals and compared with the angle of the knee joint, the extension torque and the extension speed. The extension torque was set at four levels with 10% intervals between 40 and 70% of the maximum voluntary contraction. The extension speed was set at five levels with 60 degrees /s intervals between 0 and 240 degrees /s. Because the joint angle unsystematically affected the MFCV, EMG variables at a given joint angle were extracted for comparison. The influence of the extension torque and speed on the extracted EMG variables was clarified with an ANOVA and a regression analysis. The statistical analyses showed that MFCV increased with the extension torque but did not depend on the extension speed. In contrast, MDF was independent of the extension torque but was dependent on the extension speed. MDF thus showed a behavior different from that of MFCV. It became clear that if MFCV is used as an indicator of muscular fatigue during dynamic exercises, it is at least necessary to extract MFCV at a predetermined joint angle and then remove the influence of extension torque on MFCV.     

Mitochondria in platinum resistant cells.

Based on the previous report showing that mitochondrial (MT) alteration is associated with platinum (Pt) resistance, we have determined how the alternative MT function is involved in Pt cell cytotoxicity particularly in relation to the apoptosis. MT membrane potential (delta psi m) semi-quantitatively assessed by rhodamin 123 (Rh) sensitivity was significantly elevated in acquired Pt-resistant 2008/C13*5.25 cells (C13) established from its parental 2008 cells or known intrinsic Pt-resistant JHOC cells established from ovarian clear cell adenocarcinoma. Laser confocal microscopy of these cells stained with Rh revealed that MT in Pt-resistant cells were distributed in whole cytoplasm with relatively higher fluorescent intensity whereas MT in Pt-sensitive cells were localized in perinuclear space with lower fluorescent intensity. Electron microscopy showed the predominantly condensed MT in which crestal structure was not observed clearly in Pt-resistant cells. Western blot analysis using murine monoclonal anti-Bcl-2 antibody showed more than 5-fold Bcl-2 overexpression in Pt-resistant cells in response to cisplatin treatment. Cytochrome C (CytC) in MT was released from MT into cytoplasm in response to cisplatin treatment in Pt-sensitive cells, whereas up-regulation of CytC level in MT rather than CytC release from MT was observed in Pt-resistant cells. These data are strongly suggesting that changes at MT level would impact on the relative resistance of malignant cells to undergo drug-induced apoptosis.