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The blood luteinizing hormone (LH) surge in cows is well studied. However, little is known about urinary LH in cows. This study examined urinary LH concentrations after administration of gonadotropin-releasing hormone (GnRH) in six Japanese black cows to induce LH secretion from the pituitary gland into the bloodstream. Abrupt rises in plasma and urinary LH were observed after GnRH administration. Plasma and urinary LH peaked at 2 and 5 hr, respectively. A positive correlation was observed between plasma LH concentrations and urinary LH amounts. Ovulation was confirmed in the cows after 48 hr of GnRH administration. These data strongly suggest that urinary LH is derived from plasma LH, which triggers ovulation in cows.

Dermal papilla cells (DPCs) play crucial roles in hair regeneration, but they readily lose their hair‐forming ability during in vitro culture. Although the formation of spheroids partially restores the ability, shrinkage of the spheroids makes it difficult to maintain cellular viability. To address this problem, we stimulated DPCs with factors known to induce adipogenic and/or osteogenic differentiation, because DPCs share unique gene expression profiles with adipocytes and osteocytes. We isolated DPCs from versican (vcan)–GFP mice, in which GFP is expressed under the control of a vcan promoter, which is strongly active in DPCs of anagen hair follicles. GFP fluorescence was most intense when the spheroids were made from DPCs cultured in a half‐diluted combination of adipogenic and osteogenic media (CAO1/2), a Dulbecco’s modified Eagle’s medium‐based medium that contains 10% FBS, 275 nm dexamethasone, 2.5 mm β‐glycerol phosphate, 12.5 µg·mL−1 ascorbic acid, 0.125 µm isobutylmethylxanthine and 2.5 ng·mL−1 insulin. The dose of each additive used was less than the optimal dose for adipogenic or osteogenic differentiation, and shrinkage of the spheroids was avoided through the addition of fibroblast growth factor 2 and platelet‐derived growth factor‐AA to CAO1/2. In addition, the gene and protein expression of vcan, osteopontin, alkaline phosphatase and α‐smooth muscle actin in the spheroids were augmented to levels similar to those of the intact dermal papillae, which exhibited restored hair‐forming activity. In conclusion, a combination of certain adipogenic and osteogenic inducers, together with fibroblast growth factor 2 and platelet‐derived growth factor‐AA, can promote differentiation toward the DPC lineage., Dermal papilla cells (DPCs) were isolated from versican–GFP mice. Green fluorescence (indicative of hair‐forming activity) and DPC‐specific gene expression were enhanced in spheroids derived from DPCs when they were cultured with both adipogenic and osteogenic differentiation factors at moderate concentrations in addition to fibroblast growth factor 2 and platelet‐derived growth factor‐AA (half‐diluted combination of adipogenic and osteogenic media). Hair neogenesis was observed only in nude mice grafted with these spheroids.

Basket clam soup, a popular Asian dish, is prepared by boiling clams in hot water. The soup is generally cloudy and considered more delicious as cloudiness increases. However, the identity of the whitening ingredients and their relationship with taste remain unclear. In this study, we aimed to identify the components that contribute to the white color of the boiled soup. The white component was precipitated with trichloroacetic acid and reacted positively with ninhydrin, indicating the presence of proteins. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an intense band was observed at 33 kDa. Peptide mass fingerprinting of this band using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry revealed the protein to be tropomyosin. Basket clam tropomyosin expressed and purified from Escherichia coli turned the extracted solution white, confirming that tropomyosin contributed to the white color of clam soup.

The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function.

Summary Background Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family and is involved in various diseases including cancers. Aptamers are synthetic oligonucleotides (RNA or DNA) that fold into unique three-dimensional structures and specifically bind to their targets with high affinity. We aimed to generate an aptamer with high affinity and specificity for HB-EGF. Methods Recombinant human HB-EGF (rhHB-EGF), comprised of the extracellular EGF-like and heparin-binding domains of HB-EGF, was used as the target. The aptamer against HB-EGF (the anti-HB-EGF aptamer) was obtained by systematic evolution of ligands by exponential enrichment (SELEX). Results After the 10th round of SELEX, aptamers were reverse-transcribed and PCR-amplified. Within obtained forty-six clones, twenty-three were identical (the anti-HB-EGF aptamer). The analysis using wireless-electrodeless quartz crystal microbalance revealed that the anti-HB-EGF aptamer had high affinity for rhHB-EGF (KD value: 12.2 ± 1.1 nmol/L). The dot-blot analysis revealed that the anti-HB-EGF aptamer specifically bound to rhHB-EGF. The analysis using confocal microscopy indicated that the anti-HB-EGF aptamer also bound to membrane-bound HB-EGF. Western-blot assay indicated that the anti-HB-EGF aptamer inhibited the phosphorylation of rhHB-EGF-mediated EGF receptor (EGFR). Conclusion We identified a novel RNA aptamer that bound with high affinity and specificity to rhHB-EGF and potently inhibited the rhHB-EGF-mediated phosphorylation of EGFR. The anti-HB-EGF aptamer may be a promising therapeutic agent for specifically neutralizing HB-EGF signaling.

There are few methods available for injectable liposome production under good manufacturing practices (GMP). Injectable liposome production processes under GMP generally consist of liposome formation, size homogenization, organic solvent removal, liposome concentration control and sterilization. However, these complicated and separate processes make it difficult to maintain scalability, reproducibility and sterility. To overcome these limitations, we developed a novel one-step in-line closed liposome production system that integrated all production processes by combining the in-line thermal mixing device with modified counterflow dialysis. To validate the system, we produced liposomal cyclosporine A (Lipo-CsA) and lyophilized the liposomes. The three independent pilot batches were highly reproducible and passed the quality specifications for injectable drugs, demonstrating that this system could be used under GMP. The accelerated stability test suggested that the liposomes would be stable in long-term storage. This one-step system facilitates a fully automated and unattended production of injectable liposomes under GMP.

The aim of the present study was to accomplish de novo generation of reconstituted human skin with enriched hair follicles. Dermal papillae (DP) are known to play a crucial organizing role in hair follicle induction. However, generation of enriched human hair follicles using cultured DP cells has not been accomplished because DP cells easily lose their hair-inducing ability with culturing. To enhance the hair-inducing ability of DP cells, Wnt signaling pathway activation or three-dimensional (3D) spheroid culture methods were employed in previous studies. Herein, we assessed effects of the canonical Wnt/β-catenin signaling activator CHIR99021 and found that it enhanced the expression of DP signature genes associated with hair-inducing ability. Further comparison of three different 3D culture methods revealed the highest expression of DP signature genes in spheroids generated by a floating drop method compared with other methods. CHIR99021 synergistically increased expression of DP signature genes in combination with floating drop culture. "Reconstituted skin assay" prepared using the most promising CHIR99021-stimulated 3D spheroids showed enrichment for human hair follicles. Labeled DP spheroids and derived cells were primarily found to be DP and dermal sheath cup (DSC) cells, implying organization of hair formation by DP spheroids. Finally, to evaluate the functional features of generated human skin and hair follicles, we injected human DSC cells, which reportedly show DP precursor behavior, and exhibit hair-inducing ability through incorporation into hair follicles, into mice. Histological studies revealed injected DSC cells in dermal sheath of hair follicles, consistent with a previous report, thus verifying the functionality of generated skin and hair follicles. Collectively, our findings demonstrate that DP spheroids synergistically stimulated by CHIR99021 and 3D culture contributed to hair follicle formation, thus making it possible to generate reconstituted hair follicle-enriched human skin with functional features.

Background The uterus is an organ that is directly accessible via the transvaginal route, whereas the drug delivery system and the gene delivery system (GDS) for the uterus are very limited, even in animal models. In the present study, we optimized a bionanocapsule (BNC) comprising a hepatitis B virus envelope L-protein particle, for which a structurally similar particle has been used as an immunogen of a conventional HB vaccine worldwide for more than 30 years, as a local uterine GDS using a mouse model. Methods To display various antibodies for re-targeting to different cells other than hepatic cells, the pre-S1 region of BNC was replaced with a tandem form of the protein A-derived immunoglobulin G Fc-interacting region (Z domain, ZZ-BNC). To induce strong cell adhesion after local administration into the uterine cavity, ZZ-BNC was modified with a transactivator of transcription (TAT) peptide. Results Gene transfer using TAT-modified ZZ-BNC is approximately 5000- or 18-fold more efficient than the introduction of the same dose of naked DNAs or the use of the cationic liposomes, respectively. TAT-modified ZZ-BNC was rapidly eliminated from the uterus and had no effect on the pregnancy rate, litter size or fetal growth. Conclusions TAT-modified ZZ-BNC could be a useful GDS for uterine endometrial therapy via local uterine injection.

We herein report a teenage girl who had been taking oral contraceptive pills for three months and complained of left lower abdominal pain that had continued for two months. A physical examination indicated anterior cutaneous nerve entrapment syndrome (ACNES), although no abnormality was found in various biochemical and imaging examinations. The pain was only transiently ameliorated by trigger-point injection, and neurectomy surgery was eventually effective. Sex steroids can be involved in the progress of local tissue edema causing ACNES. ACNES should be considered in cases of abdominal pain in patients taking oral contraceptives.

Rationale: Doxorubicin is an effective chemotherapeutic agent for cancer, but its use is often limited by cardiotoxicity. Doxorubicin causes endoplasmic reticulum (ER) dilation in cardiomyocytes, and we have demonstrated that ER stress plays important roles in the pathophysiology of heart failure. Objective: We evaluated the role of ER stress in doxorubicin-induced cardiotoxicity and examined whether the chemical ER chaperone could prevent doxorubicin-induced cardiac dysfunction. Methods and Results: We confirmed that doxorubicin caused ER dilation in mouse hearts, indicating that doxorubicin may affect ER function. Doxorubicin activated an ER transmembrane stress sensor, activating transcription factor 6, in cultured cardiomyocytes and mouse hearts. However, doxorubicin suppressed the expression of genes downstream of activating transcription factor 6, including X-box binding protein 1. The decreased levels of X-box binding protein 1 resulted in a failure to induce the expression of the ER chaperone glucose-regulated protein 78 which plays a major role in adaptive responses to ER stress. In addition, doxorubicin activated caspase-12, an ER membrane–resident apoptotic molecule, which can lead to cardiomyocyte apoptosis and cardiac dysfunction. Cardiac-specific overexpression of glucose-regulated protein 78 by adeno-associated virus 9 or the administration of the chemical ER chaperone 4-phenylbutyrate attenuated caspase-12 cleavage, and alleviated cardiac apoptosis and dysfunction induced by doxorubicin. Conclusions: Doxorubicin activated the ER stress–initiated apoptotic response without inducing the ER chaperone glucose-regulated protein 78, further augmenting ER stress in mouse hearts. Cardiac-specific overexpression of glucose-regulated protein 78 or the administration of the chemical ER chaperone alleviated the cardiac dysfunction induced by doxorubicin and may facilitate the safe use of doxorubicin for cancer treatment.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.

Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes.

In this study, by conducting the creep–fatigue crack growth tests using circular notched round bar specimens for Alloy 617, the crack growth behaviour was characterized on the basis of crack growth data obtained from the direct current potential drop (DCPD) method, which results in the assurance of the high accuracy of crack length measurement. Furthermore, on the basis of catastrophe theory, an attempt is made to predict theoretically the threshold temperature of creep crack growth related to the diffusion creep mechanism.

The W-added 12%Cr ferritic heat resistant steel has been developed as a turbine rotor material which is used under high temperature creep-fatigue conditions. In this study, crack growth tests using C(T) specimen under the creep-fatigue interaction conditions were conducted under various conditions of stress holding time, applied stress and temperatures. As a result, the effect of load frequency on crack growth behaviour and its life for this steel was characterised by the three-dimensional curved surface based on the concept of non-equilibrium science. Furthermore, a predictive law of crack growth life under the creep-fatigue interaction conditions was derived based on the concept of Q* with the transition function of crack growth life from fatigue to creep.

Almost all organisms maintain a circadian clock from birth to death to synchronize their own physiology and behavior with the earth’s rotation. Because the in vivo evaluation of human circadian characteristics is labor-intensive, in vitro or ex vivo approaches could provide advantages. In this study, to enable the simple and non-invasive evaluation of autonomous circadian oscillation, we established a method for monitoring clock gene expression by performing ex vivo culture of whole hair root tissue. This method is extremely simple and imposes little burden on subjects. Results obtained using Cryptochrome-deficient mice support that circadian period length in hair tissue correlates with intrinsic period length observed in physiology and behavior. We then applied this method to old-old subjects with severe dementia, who showed abnormal circadian behavior, and found that their peripheral clocks autonomously oscillated in a manner similar to those of healthy or younger subjects, indicating that the effect of cellular senescence on the autonomous clock oscillator is limited at least in some cell types. Although further validation may be required, the hair tissue-based culture assay would be a tool to investigate intrinsic circadian characteristics in humans.

Background and purpose ST2 is one of the interleukin (IL)-1 receptor family members comprising of membrane-bound (ST2L) and soluble (sST2) isoforms. Clinical trials have revealed that serum sST2 levels predict outcome in patient with myocardial infarction or chronic heart failure (HF). Meanwhile, we and others have reported that ablation of ST2 caused exaggerated cardiac remodeling in both ischemic and non-ischemic HF. Here, we tested whether IL-33, the ligand for ST2, protects myocardium against HF induced by mechanical overload using ligand specific knockout (IL-33 −/− ) mice. Methods and results Transverse aortic constriction (TAC)/sham surgery were carried out in both IL-33 and WT-littermates. Echocardiographic measurements were performed at frequent interval during the study period. Heart was harvested for RNA and histological measurements. Following mechanical overload by TAC, myocardial mRNA expressions of Th1 cytokines, such as TNF-α were enhanced in IL-33 −/− mice than in WT mice. After 8-weeks, IL-33 −/− mice exhibited exacerbated left ventricular hypertrophy, increased chamber dilation, reduced fractional shortening, aggravated fibrosis, inflammation, and impaired survival compared with WT littermates. Accordingly, myocardial mRNA expressions of hypertrophic (c-Myc/BNP) molecular markers were also significantly enhanced in IL-33 −/− mice than those in WT mice. Conclusions We report for the first time that ablation of IL-33 directly and significantly leads to exacerbate cardiac remodeling with impaired cardiac function and survival upon mechanical stress. These data highlight the cardioprotective role of IL-33/ST2 system in the stressed myocardium and reveal a potential therapeutic role for IL-33 in non-ischemic HF.

In order to detect creep–fatigue damage before the crack initiation stage, an investigation into damage behaviour based on the electron backscattered diffraction (EBSD) method for a polycrystalline nickel base superalloy has been carried out. The average misorientation within grains increases linearly up to the initiation of cracks with the increase in creep strains. Even if stress distributions exist by stress concentration, assessment in all the damage areas allowed the evaluation of creep damage regardless of geometrical influence. Furthermore, the influence of strain rates, introducing fatigue and testing temperatures are hardly observed in the misorientation analysis. Misorientation almost corresponded to inelastic strain regardless of those influences. Quite a small misorientation caused by fatigue led to an equivalent result between creep conditions and creep–fatigue conditions. It is concluded that the misorientation analysis of damaged materials based on the EBSD method allows the quantit...

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.

In this study, to clarify the behaviour of micro- and macro-creep damage progression for P92 under multiaxial stress field, interrupted creep tests, analysis of multiaxial stress and detailed the cross-sectional observations were conducted on a circular notched round bar specimen which produces the multiaxial stress field due to the plastic constraint. As a result, creep voids were initiated at the early stage and they were formed up to the final fracture. These phenomena were found to be detected using direct current potential drop (DCPD) method. These results concern the development of the measurement of creep crack initiation. The distribution of high void area fraction was in good agreement with that of high hydrostatic stress and high multiaxial stress. This result indicates that multiaxial stress affects the void formation. Furthermore, the micro-creep damage of each interrupted specimen was evaluated by using the electron backscatter diffraction (EBSD) method which can analyse crystallograp...

Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.

Although amiodarone is recognized as the most effective anti-arrhythmic drug available, it has negative hemodynamic effects. Nano-sized liposomes can accumulate in and selectively deliver drugs to ischemic/reperfused (I/R) myocardium, which may augment drug effects and reduce side effects. We investigated the effects of liposomal amiodarone on lethal arrhythmias and hemodynamic parameters in an ischemia/reperfusion rat model.We prepared liposomal amiodarone (mean diameter: 113 ± 8 nm) by a thin-film method. The left coronary artery of experimental rats was occluded for 5 min followed by reperfusion. Ex vivo fluorescent imaging revealed that intravenously administered fluorescent-labeled nano-sized beads accumulated in the I/R myocardium. Amiodarone was measurable in samples from the I/R myocardium when liposomal amiodarone, but not amiodarone, was administered. Although the intravenous administration of amiodarone (3 mg/kg) or liposomal amiodarone (3 mg/kg) reduced heart rate and systolic blood pressure compared with saline, the decrease in heart rate or systolic blood pressure caused by liposomal amiodarone was smaller compared with a corresponding dose of free amiodarone. The intravenous administration of liposomal amiodarone (3 mg/kg), but not free amiodarone (3 mg/kg), 5 min before ischemia showed a significantly reduced duration of lethal arrhythmias (18 ± 9 s) and mortality (0 %) during the reperfusion period compared with saline (195 ± 42 s, 71 %, respectively).Targeting the delivery of liposomal amiodarone to ischemic/reperfused myocardium reduces the mortality due to lethal arrhythmia and the negative hemodynamic changes caused by amiodarone. Nano-size liposomes may be a promising drug delivery system for targeting I/R myocardium with cardioprotective agents.

Reepithelialization of skin which comprises epidermis and dermis has not been fully elucidated due to the complexity of the participants as well as the interactions therein. In this study, the intrinsic roles and behaviors of epidermis itself during wound closure on neonatal rat skin were explored by developing and utilizing a novel in vivo wound model, termed “shallow incisional wound” in which the injury of dermis was minimized. The shallow wounds were closed by 12 h postwounding (PW) by the migration of the wound-marginal epidermal sheets in which activated myosin light chain (p-MLC) was predominantly detected at the lateral plasma membrane of individual cells. By local administration of Rho-associated protein kinase (ROCK) inhibitor Y27632, p-MLC disappeared at the wound margin and wounds were not closed by 12 h PW. Inhibition of Rac 1 by NSC23766 also resulted in hold of wound closure by 12 h PW, though NSC23766 somewhat slowly acted on p-MLC expression. These results suggest that, without joining of dermis, epidermal cells have a potential ability of closing wounds by active epithelial sheet movement integrated by Rho family small GTPases-dependent extension and contraction of the individual cell bodies.

Full-thickness incisional wounds were made on the dorsal skin of 1-day-old rats to elucidate the mechanism of the fluctuation of the epidermal thickness after the wound closure. The thickness of the epidermis covering the wound reached a peak around 96 h post-wounding (PW), and became thinner thereafter. The analyses of the cell proliferation and apoptosis at the epidermal wound regions revealed that the rate of TUNEL-positive cells that displays the cells undergoing apoptosis increased as the epidermis became thinner around 120 h PW. Next, immunohistochemical analyses using antibodies against keratinocyte differentiation marker proteins indicated that the delay or interruption of the spinous to granular transition from 96 to 120 h PW might result in the epidermal thickening in the wound region. Third, the region undyed with anti-caspase-14 antibody extended downward in the thickened epidermis by 96 h PW, and in turn, it became intensely and widely stained with this antibody in the thinning epidermis by 120 h PW. Taken together, it is likely that the delay and acceleration of the terminal differentiation, including cornification of the epidermal keratinocytes may coordinately cause the fluctuation of the thickness of the epidermis at the wound site in rat neonates.

Directionally solidified Ni-base superalloy is used for gas turbine blades for high efficiency thermal power plant. Since gas turbine blades are subject to high temperature creep condition due to the high speed rotation of rotor, it is important to evaluate the creep strength. There are many studies using the smooth specimen for directionally solidified Ni-base superalloy. However, these are not so many researches which concern the mechanical behavior of a notched specimen. Therefore, the researches of creep damage formation and the crack growth behavior around a notch tip have not yet been clarified. Recently, electron backscatter diffraction (EBSD) method has been conducted to evaluate the creep damage for Ni-base superalloy. However, most of the studies also use the smooth specimen. When the materials are practically used for the component of structures, creep damage or crack may be originated at the site of stress concentration of equipment such as cooling holes. Therefore, in order to evaluate the creep damage formation and the crack growth behavior, it is important to conduct the research using notched specimens from the view point of application to actual components. In this study, creep damage formation and crack growth behavior of a notched specimen for directionally solidified Ni-base superalloy CM247LC under high temperature creep condition were investigated by conducting experiment and mechanical analysis. The interrupted observational test of creep crack growth was conducted to investigate the damage formation and the crack growth behavior around notches. In addition, the In-situ observation and the metallographical investigations were conducted for creep damaged specimens using SEM / EBSD analysis. Furthermore, in order to clarify the mechanism of creep damage formation behavior, the designed two-dimensional elastic-plastic creep finite element analysis was conducted for the model with various distributed grains obtained by EBSD analysis. And this analytical results were compared with experimental results. As a result, the micro creep crack around a notch tip was found to be caused by accumulation of micro damage and voids. In addition, macro cracks were found to initiate just before final unstable fracture. However, it is necessary to take into account for the variety of mechanical properties of each crystal orientation, the designed two-dimensional elastic-plastic creep finite element analysis was found to well represent the creep damage formation observed in experiments.

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1’s cell differentiation promotion, suggesting the possibility that IGF-1’s differentiation-promotion effect is an indirect effect via IGF-1’s cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100–500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-l-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

Anaphylactoid reaction is a rapid systemic allergic reaction to many kinds of allergen. The peak age of onset is in the forties and there are not many reports on anaphylactoid reactions in pediatric patients. We report two cases of pediatric patients who underwent surgical treatment on retinoblastoma and developed anaphylactoid reaction probably caused by neostigmine. General anesthesia was induced with fentanyl, sevoflurane, dinitrogen monoxide, and rocronium. The procedure was uneventfully completed. Just after the administration of neostigmine to reverse rocronium, the patients showed red flare on the face and chest, and wheezes were heard, but the vital signs were relatively stable. The rapid onset from the administration of neostigmine to the allergic reaction accompanied by skin and respiratory manifestations strongly suggested the anaphylactoid reaction to neostigmine.