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Mesenchymal stem cells could potentially be used in the clinical treatment of muscle disorders and muscle regeneration. Adipose-derived stem cells (ADSCs) can be easily isolated from adipose tissue, as opposed to stem cells of other tissues. We believe that cell therapy using ADSCs could be applied to muscle disorders in horses and other species. We sought to improve the myogenic differentiation potential of equine ADSCs (eqADSCs) using a MyoD lentiviral vector. MyoD lentiviruses were transduced into eqADSCs and selected using puromycin. Cells were cultured in differentiation media containing 5% horse serum, and after 5 days the MyoD-transduced cells differentiated into myogenic cells (MyoD-eqADSCs). Using green fluorescent protein (GFP), MyoD-eqADSCs were purified and transplanted into the tibialis anterior muscles of mice after they were injured with the myotoxin notexin. The mice were sacrificed to examine any regeneration in the tibialis anterior muscle 4 weeks after the MyoD-eqADSCs were injected. The MyoD-eqADSCs cultured in growth media expressed murine and equine MyoD; however, they did not express late differentiation markers such as myogenin (MYOG). When cells were grown in differentiation media, the expression of MYOG was clearly observed. According to our reverse transcription polymerase chain reaction and immunocytochemistry results, MyoD-eqADSCs expressed terminal myogenic phase genes, such as those encoding dystrophin, myosin heavy chain, and troponin I. The MyoD-eqADSCs fused to each other, and the formation of myotube-like cells from myoblasts in differentiation media occurred between days 5 and 14 postplating. In mice, we observed GFP-positive myofibers, which had differentiated from the injected MyoD-eqADSCs. Our approaches improved the myogenic differentiation of eqADSCs through the forced expression of murine MyoD. Our findings suggest that limitations in the treatment of equine muscle disorders could be overcome using ADSCs.

Sauchinone is a lignan isolated from Saururus chinensis known to suppress nitric oxide (NO) activity. Previous studies demonstrate that NO plays a key role in methamphetamine-induced neurotoxicity. Thus, we hypothesized that sauchinone could have a suppressive effect on the neurotoxicity induced by methamphetamine. Repeated injections of methamphetamine cause degeneration of dopaminergic nerve terminals, whereas sauchinone treatment significantly prevented this degeneration. Sauchinone treatment also inhibited the methamphetamine-induced activation of glia cells and the production of NO via a blockade of inducible NO synthase protein expression. Our results suggest that sauchinone can prevent methamphetamine-induced neurotoxicity through the suppression of NO production. Keywords:: methamphetamine, sauchinone, nitric oxide

Isoliquiritigenin (ISL) suppresses cocaine-induced extracellular dopamine levels and has a neuroprotective effect in cocaine-treated rat brain. Here, we examine the effect of ISL on methamphetamine-induced striatal neurotoxicity. Repeated injections of methamphetamine cause the loss of striatal dopamine transporter (DAT) and tyrosine hydroxylase (TH). Intraperitoneal injection of ISL prior to methamphetamine injection significantly prevented methamphetamine-induced reduction of DAT and TH. ISL also suppressed methamphetamine-induced activation of glial cells. Moreover, ISL impeded the expression of nitric oxide synthase and the activation of NF-κB through blockage of its phosphorylation. Our results suggest that ISL protects against methamphetamine-induced neurotoxicity by inhibition of NF-κB activation. Keywords:: methamphetamine, neurotoxicity, isoliquiritigenin

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder caused by mutations in the dystrophin gene. Adipose-derived stem cells (ASCs) are an attractive source of cells for stem cell therapy. Losartan has been reported to improve ASC transplantation in injured mouse muscles. In the present study, we investigated whether the combined treatment of losartan and ASCs in the injured muscles of mdx mice improves regeneration. The combined treatment of ASCs and losartan remarkably improved muscle regeneration and induced muscle hypertrophy. In addition, ASCs and losartan treatment downregulated transforming growth factor-β and inhibited muscle fibrosis. We observed cells coexpressing green fluorescent protein (GFP) and dystrophin in the muscle samples of mice transplanted with GFP-positive ASCs. In the coculture in vitro experiment, we also observed that the GFP ASCs differentiated into dystrophin-expressing myotubes. The present study shows that the combination of transplanted ASCs and treatment with losartan ameliorated muscle fibrosis and improved muscle regeneration in injured mdx mice. Thus, we suggest that combined treatment with losartan and ASCs could help to improve muscle regeneration in the muscles of injured patients, including DMD patients.

Hepatitis C virus (HCV) has become a major public health issue. It is prevalent in most countries. HCV infection frequently begins without clinical symptoms, before progressing to persistent viremia, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) in the majority of patients (70% to 80%). Alcohol is an independent cofactor that accelerates the development of HCC in chronic hepatitis C patients. The purpose of the current study was to evaluate ethanol-induced hepatic changes in HCV core-Tg mice and mutant core Tg mice. Wild type (NTG), core wild-Tg mice (TG-K), mutant core 116-Tg mice (TG-116) and mutant core 99-Tg mice (TG-99) were used in this investigation. All groups were given drinking water with 10% ethanol and 5% sucrose for 13 weeks. To observe liver morphological changes, we performed histopathological and immunohistochemical examinations. Histopathologically, NTG, TG-K and TG-116 mice showed moderate centrilobular necrosis, while severe centrilobular necrosis and hepatocyte dissociation were observed in TG-99 mice with increasing lymphocyte infiltration and piecemeal necrosis. In all groups, a small amount of collagen fiber was found, principally in portal areas. None of the mice were found to have myofibroblasts based on immunohistochemical staining specific for a-SMA. CYP2E1-positive cells were clearly detected in the centrilobular area in all groups. In the TG-99 mice, we also observed cells positive for CK8/18, TGF-ß1 and phosphorylated (p)-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p < 0.01). Based on our data, alcohol intake induced piecemeal necrosis and hepatocyte dissociation in the TG-99 mice. These phenomena involved activation of the TGF-ß1/p-Smad2/3/p21 signaling pathway in hepatocytes. Data from this study will be useful for elucidating the association between alcohol intake and HCV infection. [ABSTRACT FROM AUTHOR]

Smad3 is a key mediator of the transforming growth factor (TGF)-ß1 signaling pathway that plays central role in inflammation and fibrosis. In present study, we evaluated the effect of Smad3 deficiency in Smad3-/- mice with carbon tetrachloride (CCl4)-induced liver fibrosis. The animals were received CCl4 or olive oil three times a week for 4 weeks. Histopathological analyses were performed to evaluate the fibrosis development in the mice. Alteration of protein expression controlled by Smad3 was examined using a proteomic analysis. CCl4-induced liver fibrosis was rarely detected in Smad3-/- mice compared to Smad3+/+. Proteomic analysis revealed that proteins related to antioxidant activities such as senescence marker protein-30 (SMP30), selenium-binding proteins (SP56) and glutathione S-transferases (GSTs) were up-regulated in Smad3-/- mice. Western blot analysis confirmed that SMP30 protein expression was increased in Smad3-/- mice. And SMP30 levels were decreased in CCl4-treated Smad3+/+ and Smad3-/- mice. These results indicate that Smad3 deficiency influences the proteins level related to antioxidant activities during early liver fibrosis. Thus, we suggest that Smad3 deteriorate hepatic injury by inhibitor of antioxidant proteins as well as mediator of TGF-ß1 signaling. [ABSTRACT FROM AUTHOR]

Recently, senescence marker protein-30 (SMP30) knockout (KO) mice have been reported to be susceptible to apoptosis, however, the role of SMP30 has not been characterized in the small intestine. The aim of the present study is to investigate the role of SMP30 in the process of spontaneous and ?-radiation-induced apoptosis in mouse small intestine. Eight-week-old male wild-type (WT) mice and SMP30 KO mice were examined after exposure to 0, 1, 3, 5, and 9 Gy of ?-radiation. Apoptosis in the crypts of the small intestine increased in the 0 to 5 Gy radiated SMP30 KO and WT mice. Radiation-induced apoptosis and the BAX/Bcl-2 ratio in the SMP30 KO mice were significantly increased in comparison to each identically treated group of WT mice (p < 0.05). The levels of spontaneous apoptosis in both WT and KO mice were similar (p > 0.05), indicating that increased apoptosis of crypt cells of SMP30 KO by irradiation can be associated with SMP30 depletion. These results suggested that SMP30 might be involved in overriding the apoptotic homeostatic mechanism in response to DNA damage. [ABSTRACT FROM AUTHOR]

Aim of study A bioactive ceramics has been reported to regulate the expression of inflammatory cytokines in macrophage cells activated by lipopolysaccharides (LPS). In present study, we investigated the anti-inflammatory effect of bioactive ceramics using liver injury model in mouse. Materials and Methods Mice were divided into three groups: Normal group, LPS group (LPS and no ceramics treatment), Ceramics group (LPS and ceramics treatment). Results LPS administration induced the increase of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse. The losses of cytoplasm of hepatocytes due to LPS caused the increase of AST and ALT in mouse plasma. In Ceramics group, however, the concentration of AST and ALT were much lower than LPS group until 6 weeks. And the losses of cytoplasm were rarely seen in Ceramics group. RT-PCR results showed that the decrease of proinflammatory cytokines such as IL-1β and IL-6 was observed in Ceramics group. Moreover, TGF-β1 and VEGF expression was increased in Ceramics group. Conclusion Bioactive ceramics effectively protected endotoxin-induced liver injury by attenuation of inflammatory processes in mice. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular injury through proliferation and migration. Pro-inflammatory cytokines and cyclooxygenase (COX)-2 and nitric oxide synthase (NOS) are highly associated with the pathogenesis of VSMCs. We investigated the effect of bioactive ceramics on the expression of inflammatory cytokines, COX-2, and inducible NOS (iNOS) induced by phorbol 12-myristate 13-acetate (PMA) in rat VSMCs. The ceramics inhibited mRNA expression of IL-1β, TNF-α, IL-6, COX-2, and iNOS. Prostaglandin release was also diminished by the ceramics. The bioactive ceramics effect on cytokines, COX-2, and iNOS expression was achieved by inhibition of NF-κB activity. Interestingly, the ceramics-induced up-regulation of expression of endothelial NOS resulted in an increase of nitric oxide production. Thus, bioactive ceramics may have dual effects on the pathogenesis of VSMCs by regulation of NF-κB activity and NO production. [ABSTRACT FROM AUTHOR]

Calcium (Ca[sup 2+]) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca[sup 2+] concentration. Members of the Ca[sup 2+]-binding proteins play a central function in the transduction of Ca[sup 2+] signals, exerting their roles through a Ca[sup 2+]-dependent interaction with their target proteins, spatially and temporally. By performing a suppression subtractive hybridization screen we identified a novel mouse gene, Scarf (skin calmodulin-related factor), which has homology to calmodulin (CaM)-like Ca[sup 2+]binding protein genes and is exclusively expressed in differentiating keratinocytes in the epidermis. The Scarf open reading frame encodes a 148-amino acid protein that contains four conserved EF-hand motifs (predicted to be Ca[sup 2+]-binding domains) and has homology to mouse CaM, human CaM-like protein, hClp, and human CaM-like skin protein, hClsp. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca[sup 2+]binding method. By Southern blot and computational genome sequence analysis, a highly related gene, Scarf2, was found 15 kb downstream of Scarf on mouse chromosome 13. The functional Scarf Ca[sup 2+]-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca[sup 2+]-mediated signaling. [ABSTRACT FROM AUTHOR]

The novel Ca2+-binding protein, Scarf (skin calmodulin-related factor) belongs to the calmodulin-like protein family and is expressed in the differentiated layers of the epidermis. To determine the roles of Scarf during stratification, we set out to identify the binding target proteins by affinity chromatography and subsequent analysis by mass spectrometry. Several binding factors, including 14-3-3s, annexins, calreticulin, ERp72 (endoplasmic reticulum protein 72), and nucleolin, were identified, and their interactions with Scarf were corroborated by co-immunoprecipitation and co-localization analyses. To further understand the functions of Scarf in epidermis in vivo, we altered the epidermal Ca2+ gradient by acute barrier disruption. The change in the expression levels of Scarf and its binding target proteins were determined by immunohistochemistry and Western blot analysis. The expression of Scarf, annexins, calreticulin, and ERp72 were up-regulated by Ca2+ gradient disruption, whereas the expression of 14-3-3s and nucleolin was reduced. Because annexins, calreticulin, and ERp72 have been implicated in Ca2+-induced cellular trafficking, including the secretion of lamellar bodies and Ca2+ homeostasis, we propose that the interaction of Scarf with these proteins might be crucial in the process of barrier restoration. On the other hand, down-regulation of 14-3-3s and nucleolin is potentially involved in the process of keratinocyte differentiation and growth inhibition. The calcium-dependent localization and up-regulation of Scarf and its binding target proteins were studied in mouse keratinocytes treated with ionomycin and during the wound-healing process. We found increased expression and nuclear presence of Scarf in the epidermis of the wound edge 4 and 7 days post-wounding, entailing the role of Scarf in barrier restoration. Our results suggest that Scarf plays a critical role as a Ca2+ sensor, potentially regulating the function of its binding target proteins in a Ca2+-de- pendent manner in the process of restoration of epidermal Ca2+ gradient as well as during epidermal barrier formation. [ABSTRACT FROM AUTHOR]

Subcutaneous leiomyosarcoma is a malignant tumor that originates from smooth muscles in the skin. Herein, we report a case of subcutaneous leiomyosarcoma that occurred in the ear pinna of a 19-month-old male Smad3+/- mouse. On gross examination, the mass extended to the whole ear auricle and obscured the ear opening. The tumor was approximately 10x10 mm in size, ill-circumscribed, and firm with superficial hemorrhage. The cut surface of the mass was grayish-white and solid with focal necrosis and hemorrhage. Histologically, spindle tumor cells with cigar-like nuclei and plump cells with round nuclei formed broad interlacing fascicles. Additionally, the tumor cells invaded adjacent muscles. Immunohistochemically, the tumor sections had positive reactions for desmine and a-SMA and were partially positive for vimentin while negative for S-100 and myogenin. Based on the results of the examination, we diagnosed this case as subcutaneous leiomyosarcoma. [ABSTRACT FROM AUTHOR]