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It is hard to supply satellite cells as a cell source for therapy of muscle degenerative disease since the sampling of muscle tissue is very invasive to a patient with muscular disease. Direct conversion allows us to get specific cell types by transduction of defined transcriptional factors. To induce myogenic direct conversion, we transduced five transcriptional factors including Pax3, Sox2, Klf4, c-Myc, and Esrrb into mouse embryonic fibroblasts. We found that the transduction of the five transcriptional factors induced myogenic direct conversion of fibroblast. We revealed that the transduced cells with the five transcriptional factors were converted to myogenic lineage cells through a paraxial mesoderm-like stage. The expression level of myogenic-related genes of the transduced cells gradually increased as the passage increased. The induced myogenic lineage cells differentiated into muscle fibers in virto and in vivo. The current study revealed that the five transcription factors generated myogenic lineage cells from fibroblast via a paraxial mesoderm stage. The induced myogenic lineage cells may have a potential being applied as cell source for degenerative muscle disease.

Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.

A 7 years old, female and neutered ferret ( Mustela putorius furo ) was presented to local animal hospital due to lethargy, anorexia and hypothermia. Radiography, ultrasonography and blood test were performed. On the basis of clinical signs and tests, this case was presumed to be lymphoma. In consid-eration of this ferret’s condition, chemotherapy was carried out for the treatment. However, the ferret died a few days. After necropsy, metastatic tumors were observed over abdomen. All tumors were packed with homologous lymphocytes with pleomorphic changes and numerous mitotic figures. Consequently, this masses were diagnosed as B-cell gastrointestinal lymphoma, which were negative for CD3 on immunohistochemistry. Key words : Ferret, lymphoma, B-cell, Gastrointestinal form, Metastasis 서 론 림프종은 부신 피질 종양, 인슐린종와 더불어 페렛( Mustela putorius furo )에서 주로 발생하는 종양 중 하나다. 페렛에서 림프종이 흔히 나타나는 종양(11.9∼29.2%)이나, 정확하고 빠르게 진단하는 것이 어렵고, 다른 질병을 진단하거나 신체검사를 하는 도중에 우연히 발견되는 경우가 많다(Antinoff 와 Hahn, 2004). 림프종이 발생한 페렛에 대한 후향적 연구에 따르면 중성화된 페렛이 전체 중에 60%를 차지하고, 모발색에 따라 나열하면, fitch coat (63%), albinotic (10%), chocolate (6%), silver (5%), siamese (3%), panda (3%) 순서로 림프종이 관찰되었다고 한다(Erdman 등, 1996). 림프종은 발생 부위에 따라 발생빈도가 다르고 mul-ticentric form (40%), gastrointestinal form (45%), me-diastinal form (10%) 그리고 cutaneous form (5%)로 분류된다. 림프종 발생이 시작된 나이에 따라 장기 분포나 진행 양상이 달라진다(Onuma 등, 2008). 본 증례에서 7살 중성화된 암컷 페렛에서 전신적으로 발생한 림프종의 임상학적 그리고 병리학적 특징에 대해 살펴보고자 한다.

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.

Adrenomedullin (AM) is a peptide expressed in all body tissues, and its related receptors are increased in liver fibrosis. In this study, we evaluated the effect of AM deficiency on liver fibrogenesis induced by CCl 4 using AM heterozygous (HT) mice. The animals received a single injection of CCl 4 or olive oil for the acute experiment, and received CCl 4 or olive oil three times a week for 6 weeks for the chronic experiment. Fibrosis was accessed using histopathological analysis and the western blot. The AM HT mice showed mild pericentrilobular degeneration when compared to the AM wild type (WT) mice. In the acute experiment, there was no significant difference between the AM WT and AM HT mice. However, in the chronic experiment, the CCl 4 -treated AM HT mice showed more severe liver fibrosis than that of the CCl 4 -treated AM WT mice. The AST and ALT levels of the AM HT CCl 4 group were higher than those of the AM WT CCl 4 group. Additionally, the collagen deposition, α-SMA protein and TGF-β protein were increased in the AM HT CCl 4 group when compared to the AM WT CCl 4 group. The AM HT mice also exhibited severe lipid peroxidation through the GSH decrement. Taken together, our data suggest that AM deficiency increases the susceptibility to liver fibrosis induced by CCl 4 , indicating a novel therapeutic target for patients with liver fibrosis.

The capacity for liver regeneration involves a variety of nutritional factors. Vitamin C has multiple metabolic and antioxidant functions. In this study, we investigated the role of vitamin C in liver regeneration following hepatectomy in senescence marker protein (SMP)-30 knockout (KO) mice. Partial hepatectomy was performed by resecting the median and left lateral lobes of mice. Vitamin C accelerated liver recovery in SMP30 KO mice treated with vitamin C (KV). The livers of the KV mice exhibited lower levels of aspartate aminotransferase and lower injury than those of the KO mice. Increased type II transforming growth factor-β receptor (TGF-βRII)-mediated regeneration signaling was accompanied by HGF and cMet in the KV but not the KO mice. Consistent with this, the expression of cell cycle regulatory proteins, including cyclin D1 and proliferating cell nuclear antigen (PCNA), increased rapidly in the KV mice. Enhanced activation of ERK and GSK-3β proteins and a significantly increased number of binuclear hepatocytes were also detected in the livers of the KV mice. Moreover, the KV mice synthesized the highest levels of albumin. These data suggest that treating SMP30 knockout mice with vitamin C resulted in earlier recovery and liver regeneration by activation of the regeneration system.

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

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 α-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-β1 and phosphorylated (p)-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p &lt, 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-β1/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.

Smad3 is a key mediator of the transforming growth factor (TGF)-1 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-1 signaling.

Sauchinone is a phytochemical known as a nitric oxide (NO) inhibitor. NO is a kind of neurotransmitter and involved in psychotic effect of abuse drug. In present, we carried out a study on the effect of sauchinone on methamphetamine-induced alteration of behavior in mice. Locomotory activity and conditioned place preference (CPP) were used to evaluate behavioral changes. As a result, sauchinone inhibited the methamphetamine-induced hyperlocomotion in dose-dependent manner, whereas sauchinone had no effect on normal locomotory activity. The inhibitory effect of sauchinone on methamphetamine-induced hyperlocomotion was reversed by treatment of molsidomine, a NO donor. Sauchinone also significantly blocked the acquisition and expression of CPP induced by methamphetamine in mouse. However, it did not produce place preference or place aversion, when it was treated alone in animals. Taken together, sauchinone blocked drug reward-related behavior as well as acute hyperlocomotion induced by methamphetamine treatment.

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 gamma-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 gamma-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.

Activated microglial cells play an important role in inflammatory responses in the central nervous system (CNS) that are involved in neurodegenerative diseases. Sauchinone has been shown to modulate the expression of inflammatory factors through nuclear factor-kappa B (NF-κB) signaling pathway. Here, we examined the effect of sauchinone on the inflammatory responses of microglia cells induced by lipopolysaccharide (LPS) and explored the mechanism underlying action of sauchinone. BV2 cells treated with LPS showed an up-regulation of nitric oxide (NO) and prostaglandin (PGE(2)) release, whereas sauchinone suppressed this up-regulation. Sauchinone inhibited both mRNA and protein expression of COX-2, iNOS, TNF-α and IL-1β. In addition, sauchinone blocked the activation of NF-κB through its inhibition of I-κB phosphorylation. Interestingly, sauchinone had no effect on the LPS-induced phosphorylation of mitogen activated protein kinases (MAP kinases; ERK1/2, p38, JNK), but it did inhibit Akt phosphorylation. These results suggest that the inhibitory effect of sauchinone on the LPS-induced production of inflammatory mediator in BV2 cells is associated with the suppression of the NF-κB and Akt signaling pathways. Therefore, sauchinone may be a useful treatment for neurodegenerative disease by inhibiting inflammatory responses in activated microglia.

In the present study, functional roles of GABA receptors in the nucleus accumbens on morphine self-administration behavior were investigated. Male Sprague-Dawley rats were trained to press lever for morphine (0.1 mg/kg per infusion) during daily 1-h self-administration session. After establishing stable baseline responses, rats were given microinjections of the GABA(A) receptor agonist muscimol (0, 250 and 500 ng/microl, bilateral) or the GABA(B) receptor agonist baclofen (0, 100 and 250 ng/microl, bilateral) into the nucleus accumbens immediately before the morphine self-administration. Microinjection of muscimol (250 and 500 ng/microl) into the nucleus accumbens, but not baclofen, decreased morphine self-administration responses. These results suggest that activation of GABA(A) receptors, but not GABA(B) receptors, in the nucleus accumbens plays a critical role in modulating the reinforcing effects of morphine.

Isoliquiritigenin (ISL) is a licorice flavonoid with chalcone structure and is an active component of the root of the plant genus Glycyrrhiza. In addition to anti-inflammatory and antioxidant effects, ISL was previously reported to antagonize increased striatal dopamine release. In the present study, we aimed to investigate whether ISL has an effect on hyperlocomotion in animals subjected to acute cocaine administration and whether ISL modulates acute cocaine-induced molecular changes. To achieve our goals, we analyzed behavior and differential proteomic changes between ISL and vehicle in acute cocaine treated rats. Locomoter activity was reduced in ISL treated animals compared to vehicle in acute cocaine treated rats. Two dimensional electrophoresis (2-DE) revealed that 56 proteins were differentially expressed in response to ISL. Further proteomic analyses using mass spectroscopy, and subsequent validation experiments confirmed that ISL induced changes in proteins related to metabolism, signal transduction, protein folding and transport, oxidative stress, and neural toxicity. Furthermore, cocaine-induced neuronal toxicity was attenuated by ISL treatment, suggesting a neuroprotective role of ISL.

In our previous study we demonstrated that acupuncture at Shenmen (HT7) points suppressed a decrease of accumbal dopamine (DA) release in ethanol-withdrawn rats. Furthermore, here we found that it inhibited behavioral withdrawal signs of ethanol. In an effort to better understand the mechanisms underlying this inhibition, the potential role of GABA receptor system in acupuncture was investigated. Male Sprague-Dawley rats were treated with 3g/kg/day of ethanol (20%, w/v) or saline by intraperitoneal injection for 21 days. Following 48 or 72h of ethanol withdrawal, acupuncture was applied at bilateral HT7 for 1min. The selective GABA(A) antagonist bicuculline and the selective GABA(B) antagonist SCH 50911 were injected intraperitoneally 20min before acupuncture, respectively. Importantly, suppressive effects of acupuncture on DA deficiency were completely abolished by SCH 50911, but not by bicuculline, whereas ameliorating effects of acupuncture on ethanol withdrawal syndrome were completely blocked either by SCH 50911 or bicuculline. These results suggest that acupuncture at specific acupoint HT7 may normalize the DA release in the mesolimbic system and attenuate withdrawal syndrome through the GABA(B) receptor system in ethanol-withdrawn rats.

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-1beta, TNF-alpha, 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-kappaB 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-kappaB activity and NO production.

TGF-beta1 has been known as an important factor in tubulointerstitial fibrosis which is a common process in most progressive renal diseases. We hypothesized that the interstitial fibrosis could be prevented by abolishing TGF-beta1 function in unilateral ureteral obstruction (UUO)-induced renal fibrosis. shRNA vectors were generated to suppress TGF-beta1 expression at a high glucose concentration which allowed the maximal induction of TGF-beta1 in primary rat mesangial cells. An shRNA vector, designated shTB1d, significantly suppressed TGF-beta1 in both transcriptional and translational levels in vitro cultured cells and in vivo fibrosis-induced mouse kidney, accompanied by the suppression of target genes (e.g., type I collagen and PAI-1) of TGF-beta1. Furthermore, the shTB1d suppressed the expression of TGF-beta1 and type I collagen in tubulointerstitial cells until day 7 after UUO-induced fibrosis, but none- or vector-treated mice maintained their expression, suggesting that the TGF-beta1 shRNA delays the process of renal fibrosis in UUO mouse model. This work would provide a valuable tool to prevent tubulointerstitial fibrosis using RNA interference strategy.

Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-beta1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-beta1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-beta1 siRNAs reduced the expression of TGF-beta1 in the livers of CCl(4) injection compared with those of control group, and the expression of type I collagen and alpha-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-beta1 siRNAs inhibit TGF-beta1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human.

Calcium (Ca2+) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca2+ concentration. Members of the Ca2+-binding proteins play a central function in the transduction of Ca2+ signals, exerting their roles through a Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca2+-mediated signaling.

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

Helicobacter (H.) pylori strains that express the cagA and s1a vacA genes are associated with an increased risk for gastric cancer. Here, we examined the association between the products of these virulence genes with the development of gastric cancer by immunohistochemical staining of gastric biopsy specimens taken from 208 routine gastroscopies and 43 gastric cancer patients. The correlation was analyzed by multivariate logistic regression. CagA and VacA expressions in gastric mucosa were significantly associated with chronic gastritis (CG) and intestinal metaplasia (IM), respectively, accompanying CG independent of age. The association of CagA expression with IM accompanying CG was increased in patients over 50-year old (p < 0.01) and that of VacA with CG was significant in patients younger than 50 year (p < 0.05). VacA and CagA were associated with mild IM incidence (p = 0.025 and p = 0.076, respectively) but not advanced IM. In the 43 gastric cancer patients, positivity for VacA was significantly higher in cases of CG and IM than carcinoma (p = 0.042), while that for CagA was slightly higher for individuals with carcinoma than those with CG and IM. These results indicate that CagA and VacA are critical factors for inducing CG and the subsequent progression of IM from CG with an increasing age.

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100 % survival rate). In the non-vitamin C group, the 10 % ENA-A-treated mice were alive at week 18. The control and 5 % ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12more » putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.« less

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

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.

Glycyrrhizae radix (licorice) comprises a variety of flavonoids as major constituents including isoliquiritigenin, liquiritin, liquiritigenin, and glycyrrihizin. It has shown various biological activities such as anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to drug addiction, we carried out a study on the effect of G. radix and its active component, isoliquiritigenin, on acute cocaine-induced extracellular dopamine release in moving rats. Male Sprague-Dawley rats were orally administered with methanolic extracts of G. radix or isoliquiritigenin 1 h prior to an injection of cocaine (20 mg/kg, intraperitoneal (i.p.)). Extracellular dopamine was measured by in vivo microdialysis. Extract of G. radix and isoliquiritigenin inhibited cocaine-induced extracellular dopamine level in the nucleus accumbens by dose-dependent manner. Inhibition of dopamine release by isoliquiritigenin resulted in attenuation of the expression of c-Fos, an immediately early gene induced by cocaine. Effect of isoliquiritigenin was completely prevented by a GABA(B) receptor antagonist. Thus, these results showed that G. radix and isoliquiritigenin inhibit cocaine-induced dopamine release by modulating GABA(B) receptor, suggesting that isoliquiritigenin might be effective in blocking the reinforcing effects of cocaine.

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+-dependent manner in the process of restoration of epidermal Ca2+ gradient as well as during epidermal barrier formation.

Bioactive materials have previously been used to coat implants. In a new development for bioactive materials, a silica–ceramic mixture was found to alleviate pain (Lee, Poster presented at the Ninth World Congress of Gynecological Endocrinology, Hongkong, 2001. Poster session (p47)). Here, we hypothesized that silica–ceramic can reduce the expression and activity of cyclooxygenase 2 (COX2) or cytokines associated with inflammation. The production of COX2 and proinflammatory cytokines was investigated by reverse transcriptase (RT)-PCR and ELISA assay in macrophages stimulated by lipopolysaccharide (LPS). Silica–ceramic had no effect of COX2 expression and prostaglandin production in macrophages. However, silica–ceramic suppressed the synthesis of cytokines involved in inflammation, in particular, the expression of IL-1β and IL-6 was reduced at the transcriptional and translational levels. The involvement of NF-κB in the suppression of cytokines by silica–ceramic was examined by luciferase reporter assay. The NF-κB activity stimulated by LPS was inhibited by 20–60% with silica–ceramic compared with treatment with LPS alone. We suggest that inhibition of NF-κB activity by silica–ceramic might cause the attenuation of proinflammatory cytokine expression in macrophages. In conclusion, silica–ceramic could be an alternative approach to regulate the inflammation process. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2007

Herpes simplex virus (HSV) infection usually occurs in immunocompromised or severely debilitated patients. It is not so common in patients with renal transplants. The diagnosis can only be made histologically. It usually occurs during or shortly after treatment of graft rejection with high-dose steroids. We have recently experienced a case of HSV esophagitis and nephropathy in the renal allograft biopsy, which was identified by histology, immunostaining, and electron microscopy. A 43-year-old woman underwent cadaveric renal transplantation with cyclosporine and prednisolone treatment. Twelve months later, she developed renal insufficiency and proteinuria. Allograft renal biopsy showed some evidence of acute rejection. She was treated with 3 successive days of methylprednisolone (1.0 g/d) intravenously and continued tapering of steroids. Three weeks after steroid pulse therapy, she had throat pain, oral cavity ulcer, dysphagia, and febrile sensation. Esophagoscopy revealed multiple confluent ulcers in the whole esophagus, and biopsy showed enlarged epithelial cells with prominent nuclei. Immunohistochemically, the epithelial cells were positive with a monoclonal antibody to HSV type 1. She was started on acyclovir intravenously, which was continued for a week. After a week, her symptoms began to improve and repeat endoscopy showed no residual esophagitis. A renal allograft infection with HSV can persist in heavily immunosuppressed patients with recurrent rejection episodes. HSV mainly affects tubular cells causing necrosis, a major reason for functional deterioration. A biopsy is required for diagnosis.

During the process of epidermal differentiation, intracellular and extracellular calcium (Ca++) concentrations induce an array of signaling pathways [Berridge, M.J., Lipp, P., Bootman, M.D., 2000. The versatility and universality of calcium signaling. Nature Rev. Mol. Cell. Biol. 1, 11–21] . Keratinocytes follow a complex Ca++-dependent program of differentiation moving from the basal proliferative layer, through the spinous and granular differentiated layers to ultimately culminate in the formation of the cornified layer of the epidermis. Members of the Ca++-binding proteins play a central role in the transduction of Ca++ signals. Utilizing a suppressive subtractive hybridization screen comparing basal and differentiated keratinocytes, we identified the novel Ca++-binding protein genes, Scarf (skin Calmodulin-related factor) and Scarf2, which have homology to calmodulin (CaM). In this study, we present a comprehensive analysis of the expression pattern for Scarf and Scarf2 transcripts and proteins in the developing mouse. To examine Scarf2 expression during embryogenesis, we performed in situ hybridization, and detected expression in the hair follicle, skin and nasal epithelium. These results showed substantial overlap with the previously reported Scarf gene expression [Hwang, M., Morasso, M.I., 2003. The novel murine Ca2+-binding protein, Scarf, is differentially expressed during epidermal differentiation. J. Biol. Chem. 278, 47827–47833]. Comparing the expression patterns of Scarf and Scarf2 proteins in neonatal and adult mouse skin with several structural epidermal proteins, i.e. keratin 14 (K14), keratin 1 (K1), loricrin (LOR) and filaggrin (FIL) showed that their expression overlaps K1, an early marker of keratinocyte differentiation. Interestingly, Scarf and Scarf2 were also detected in the tongue and oral epithelia, rib bone undergoing ossification and in the medullar region of thymus.

Transmission electron microscopy of an Acanthamoeba isolate (KA/L5) from a contact lens case revealed bacterial endosymbionts within cytoplasm of the amoebae. The Acanthamoeba isolate belonged to the morphological group II. Based on the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of 18S ribosomal RNA coding DNA (rDNA), the isolate was identified as A. lugdunensis. Strain typing by isoenzyme analysis using isoelectric focusing (IEF) and mitochondrial (Mt) DNA RFLP revealed that the isolate was closely related with KA/L1, the most predominant type of isolates from contact lens storage cases, KA/E2, a clinical isolate, KA/W4, previously reported to host endosymbionts, and L3a strains of A. lugdunensis. The endosymbionts were similar to those of KA/W4 in aspects that they were randomly distributed in both trophozoites and cysts, and were rod-shaped bacteria measuring approximately 1.38 x 0.50 microns. But the number of endosymbionts per amoeba was significantly lower than that of KA/W4. They were neither limited by phagosomal membranes nor included in lacunaelike structure.