Promoting the Aging Process and Enhancing the Production of Antioxidant Components of Garlic through Pulsed Electric Field Treatments.

Shortening the aging duration and enhancing the functional components of garlic present significant technical challenges that need to be addressed. Thus, this study aimed to evaluate the potential role of pulsed electric field (PEF) treatment, a novel nonthermal food processing method, in promoting and enhancing the functional attributes of aged garlic. Our results showed that 2-4 kV/cm PEF pretreatment increased S-allyl cysteine (SAC), total polyphenol (TPC), and flavonoid contents (TFC) compared with un-pretreated garlic during aging. The browning and texture-softening were also significantly improved during processing time, though the latter showed no significant difference from the eighth day to the end of the aging process. The principal component analysis results showed that PEF positively affects the SAC and TFC formations without adverse effects. Among the PEF pretreatments, 3 kV/cm is the most effective in enhancing functional component production compared with the other PEF pretreatments. Therefore, PEF pretreatment is a time-saving process that promotes and enhances the functionality of aged garlic.

Gastroprotective effects of Machilus zuihoensis Hayata bark against acidic ethanol-induced gastric ulcer in mice.

Background and aim: In traditional medicine, Machilus zuihoensis Hayata bark (MZ) is used in combination with other medicines to treat gastric cancer, gastric ulcer (GU), and liver and cardiovascular diseases. This study aims to evaluate the gastroprotective effects and possible mechanism(s) of MZ powder against acidic ethanol (AE)-induced GU and its toxicity in mice. Experimental procedure: The gastroprotective effect of MZ powder was analyzed by orally administering MZ for 14 consecutive days before AE-inducing GU. Ulcer index (UI) and protection percentage were calculated, hematoxylin and eosin staining and periodic acid-Schiff staining were performed, and gastric mucus weights were measured. The antioxidative, anti-inflammatory, and anti-apoptotic mechanisms, and possible signaling pathway(s) were studied. Results and conclusion: Pretreatment with MZ (100 and 200 mg/kg) significantly decreased 10 µL/g AE-induced mucosal hemorrhage, edema, inflammation, and UI, resulted in protection percentages of 88.9% and 93.4%, respectively. MZ pretreatment reduced AE-induced oxidative stress by decreasing malondialdehyde level and restoring superoxide dismutase activity. MZ pretreatment demonstrated anti-inflammatory effects by reducing both serum and gastric tumor necrosis factor-α, interleukin (IL)-6, and IL-1ß levels. Furthermore, MZ pretreatment exhibited anti-apoptotic effect by decreasing Bcl-2 associated X protein/B-cell lymphoma 2 ratio. The gastroprotective mechanisms of MZ involved inactivations of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and mitogen activated protein kinase (MAPK) signaling pathways. Otherwise, 200 mg/kg MZ didn't induce liver or kidney toxicity. In conclusion, MZ protects AE-induced GU through mucus secreting, antioxidative, anti-inflammatory, and anti-apoptotic mechanisms, and inhibitions of NF-κB and MAPK signaling pathways.

Effects of Cordyceps militaris fermentation products on reproductive development in juvenile male mice.

Cordyceps militaris (CM) is a popular medicinal fungus; however, few studies have focused on its impact on the male reproductive system. We evaluated the effects of CM fermentation products on the reproductive development of juvenile male (JM) mice. Mice were divided into four experimental groups, each fed 5% CM products (weight per weight (w/w) in normal diet): extracellular polysaccharides (EPS), fermentation broth (FB), mycelia (MY), and whole fermentation products (FB plus MY, FBMY) for 28 days, while mice in the control group (CT) were fed a normal diet. Basic body parameters, testicular structure, sperm parameters, and sex hormones concentrations were analyzed. Compared to the CT group, mice in the EPS, MY, and FBMY groups showed a significantly increased mean seminiferous tubule area (p < 0.05), mice in the FB and MY groups had significantly higher sperm concentrations (p < 0.05), and mice in the EPS, FB, and FBMY groups showed significantly increased ratios of motile sperm (p < 0.05). Meanwhile, EPS significantly promoted the ability of JM mice to synthesize testosterone (p < 0.05). Furthermore, all CM products significantly increased the food intake of JM mice (p < 0.05) but did not significantly change their water intake and body weight gain (p > 0.05). In conclusion, CM products, especially EPS, exhibit strong androgen-like activities that can promote male reproductive development.

Reduction of 3-Deoxyglucosone by Epigallocatechin Gallate Results Partially from an Addition Reaction: The Possible Mechanism of Decreased 5-Hydroxymethylfurfural in Epigallocatechin Gallate-Treated Black Garlic.

5-Hydroxymethylfurfural (5-HMF) is a harmful substance generated during the processing of black garlic. Our previous research demonstrated that impregnation of black garlic with epigallocatechin gallate (EGCG) could reduce the formation of 5-HMF. However, there is still a lack of relevant research on the mechanism and structural identification of EGCG inhibiting the production of 5-HMF. In this study, an intermediate product of 5-HMF, 3-deoxyglucosone (3-DG), was found to be decreased in black garlic during the aging process, and impregnation with EGCG for 24 h further reduced the formation of 3-DG by approximately 60% in black garlic compared with that in the untreated control. The aging-mimicking reaction system of 3-DG + EGCG was employed to determine whether the reduction of 3-DG was the underlying mechanism of decreased 5-HMF formation in EGCG-treated black garlic. The results showed that EGCG accelerated the decrease of 3-DG and further attenuated 5-HMF formation, which may be caused by an additional reaction with 3-DG, as evidenced by LC-MS/MS analysis. In conclusion, this study provides new insights regarding the role of EGCG in blocking 5-HMF formation.

N-Acetyl Cysteine Overdose Inducing Hepatic Steatosis and Systemic Inflammation in Both Propacetamol-Induced Hepatotoxic and Normal Mice.

Acetaminophen (APAP) overdose induces acute liver damage and even death. The standard therapeutic dose of N-acetyl cysteine (NAC) cannot be applied to every patient, especially those with high-dose APAP poisoning. There is insufficient evidence to prove that increasing NAC dose can treat patients who failed in standard treatment. This study explores the toxicity of NAC overdose in both APAP poisoning and normal mice. Two inbred mouse strains with different sensitivities to propacetamol-induced hepatotoxicity (PIH) were treated with different NAC doses. NAC therapy decreased PIH by reducing lipid oxidation, protein nitration and inflammation, and increasing glutathione (GSH) levels and antioxidative enzyme activities. However, the therapeutic effects of NAC on PIH were dose-dependent from 125 (N125) to 275 mg/kg (N275). Elevated doses of NAC (400 and 800 mg/kg, N400 and N800) caused additional deaths in both propacetamol-treated and normal mice. N800 treatments significantly decreased hepatic GSH levels and induced inflammatory cytokines and hepatic microvesicular steatosis in both propacetamol-treated and normal mice. Furthermore, both N275 and N400 treatments decreased serum triglyceride (TG) and induced hepatic TG, whereas N800 treatment significantly increased interleukin-6, hepatic TG, and total cholesterol levels. In conclusion, NAC overdose induces hepatic and systemic inflammations and interferes with fatty acid metabolism.

The Anti-inflammatory Effects of the Bioactive Compounds Isolated from Alpinia officinarum Hance Mediated by the Suppression of NF-kappaB and MAPK Signaling.

This study was designed to evaluate the anti-inflammatory effects of Alpinia officinarum Hance extract (AOE) and identify its main active ingredients. AOE was obtained using a 95% ethanol extraction method. Lipopolysaccharide (LPS) were used to induce an inflammatory response in RAW264.7 cells. The results showed that AOE exerts anti-inflammatory effects via inhibition of prostaglandin E2 secretion and cyclooxygenase -2 (COX-2) production. We further analyzed the components of AOE using high-performance liquid chromatography and found that AOE is comprised of several bioactive flavonoids including quercetin (Q), kaempferol (K), galangin (G), and curcumin (C). These four flavonoids effectively inhibited nitric oxide (NO), interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α production. Moreover, they reduced COX-2 and inducible NO synthase expressions via regulation of nuclear factor kappa-light-chain-enhancer of activated B cells and c-Jun N-terminal kinase signaling pathways. Furthermore, we compared and contrasted the anti-inflammatory effects and mechanisms of these four flavonoids at the same dose in the LPS-induced cell inflammation model. The results showed that C is the most effective inhibitor of LPS-induced NO production. However, only Q and K effectively attenuated LPS-induced extracellular signal-regulated kinase and p38 elevations. In conclusion, AOE and its major bioactive compounds exert anti-inflammatory effects on LPS-induced inflammation. As A. officinarum Hance is much cheaper than any of its four flavonoids, especially G, we suggest using AOE as an anti-inflammatory agent.

The Capability of O-Acetyl-ADP-Ribose, an Epigenetic Metabolic Small Molecule, on Promoting the Further Spreading of Sir3 along the Telomeric Chromatin.

O-acetyl-ADP-ribose (AAR) is a metabolic small molecule relevant in epigenetics that is generated by NAD-dependent histone deacetylases, such as Sir2. The formation of silent heterochromatin in yeast requires histone deacetylation by Sir2, structural rearrangement of SIR complexes, spreading of SIR complexes along the chromatin, and additional maturation processing. AAR affects the interactions of the SIR-nucleosome in vitro and enhances the chromatin epigenetic silencing effect in vivo. In this study, using isothermal titration calorimetry (ITC) and dot blotting methods, we showed the direct interaction of AAR with Sir3. Furthermore, through chromatin immunoprecipitation (ChIP)-on-chip and chromatin affinity purification (ChAP)-on chip assays, we discovered that AAR is capable of increasing the extended spreading of Sir3 along telomeres, but not Sir2. In addition, the findings of a quantitative real-time polymerase chain reaction (qRT-PCR) and examinations of an in vitro assembly system of SIR-nucleosome heterochromatin filament were consistent with these results. This study provides evidence indicating another important effect of AAR in vivo. AAR may play a specific modulating role in the formation of silent SIR-nucleosome heterochromatin in yeast.

Stabilization of Sir3 interactions by an epigenetic metabolic small molecule, O-acetyl-ADP-ribose, on yeast SIR-nucleosome silent heterochromatin.

In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.

Enhancer role of a native metabolite, O-acetyl-ADP-ribose, on the Saccharomyces cerevisiae chromatin epigenetic gene silencing.

To study the epigenetic gene silencing, yeast is an excellent model organism. Sir proteins are required for the formation of silent heterochromatin. Sir2 couples histone deacetylation and NAD hydrolysis to generate an endogenous epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). AAR is involved in the conformational change of SIR complexes, modulates the formation of SIR-nucleosome preheterochromatin and contributes to the spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin regions. Here, we show that AAR is capable of enhancing the chromatin silencing effect under either an extra exogenous AAR or a defect AAR metabolic enzyme situation, but decreasing the chromatin silencing effect under a defect AAR synthetic enzyme state. Our results provide an evidence of biological function importance of AAR. It is indicated that AAR does not only function in vitro but also play a role in vivo to increase the effect of heterochromatin epigenetic gene silencing. However, further investigations of AAR are warranted to expand our knowledge of epigenetics and associated small molecules.

Enzymatic hydrolysates obtained from Trametes versicolor polysaccharopeptides protect human skin keratinocyte against AAPH-induced oxidative stress and inflammatory.

BACKGROUND: Polysaccharopeptides (PSPs) extracted from Trametes versicolor show antitumor, anti-inflammatory, and immunomodulation effects. According to our previous report, the enzymatic hydrolysates obtained from T versicolor PSPs by 80 U/mL ß-1,3-D-glucanase (PSPs-EH80) did not change the functional groups of PSPs but enhanced their antioxidative activities. However, the mechanism elevating the antioxidant and anti-inflammatory effect of PSPs-EH80 is not clear. AIMS: This research focused on the protective mechanism(s) of PSPs-EH80 against free radical and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative damage in human keratinocyte (HaCaT) cells. METHODS: We evaluated the anti-inflammatory potential of PSPs-EH80 by assessing its free radical-induced oxidative damage. Using the HaCaT cell as the experimental system, we tested the protective effects of PSPs-EH80 on a model of AAPH-induced cellular oxidative damage through the assessment of cell survival rate. Heme oxygenase 1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), cyclooxygenase-2 (COX-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were determined using MTT assays and Western blotting. RESULTS: We demonstrated that PSPs-EH80 significantly enhanced keratinocyte viability, and augmented the antioxidant HO-1 expressions through upregulation of the Nrf2, compared with PSPs. Furthermore, PSPs-EH80 significantly reduced AAPH-induced COX-2 expressions through downregulation of the ERK, p38, and NF-κB signaling pathways. CONCLUSION: The PSPs-EH80 exhibits a stronger antioxidant and anti-inflammatory capacity than PSPs. Therefore, PSPs-EH80 could be effective for attenuating free radical-induced oxidative damage in human skin and can be applied widely in the fields of cosmetics and medicine.

Safety and efficacy of tyrosinase inhibition of Paeonia suffruticosa Andrews extracts on human melanoma cells.

INTRODUCTION: The development of tyrosinase inhibitors is a hot research topic. Recently, the Chinese herb Paeonia suffruticosa Andrews, commonly named as Cortex Moutan (CM), was reported as being capable of reducing melanogenesis. We developed an A2058 human melanoma cell model to test the safety and efficacy of tyrosinase inhibition. The aim was to further clarify the bioactivities of CM extracts and paeonol for the purpose of skin whitening. METHODS: The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total polyphenol and flavonoid contents, and in vitro tyrosinase inhibitory effects of water and ethanol CM extracts were determined. Cellular inhibitions of tyrosinase and melanin production were also evaluated. RESULTS: Water and ethanol CM extracts were both shown to have strong DPPH scavenging abilities in a dose-dependent manner. The polyphenol content was higher in the ethanol CM extract compared to the water extract, while the flavanone content was comparable. Kinetic analyses revealed that the ethanol CM extract and paeonol are noncompetitive tyrosinase inhibitors. The cellular melanin content and l-DOPA oxidation assays demonstrated that the ethanol CM extract was an appropriate alternative whitening agent to paeonol and arbutin in ultraviolet-induced A2058 human melanoma cells. CONCLUSION: The results of this study suggest that a human cell model is more suitable for determining tyrosinase activity than mouse cell models for determining cellular tyrosinase activity and melanin production. The ethanol CM extract was also confirmed as a promising ingredient in sun protection and skin whitening cosmetics. Future work should focus on melanogenesis-related gene expressions.

Anti-Fatigue, Antioxidation, and Anti-Inflammatory Effects of Eucalyptus Oil Aromatherapy in Swimming-Exercised Rats.

Eucalyptus globulus possesses important pharmacological activities, including antioxidant and anti-inflammatory effects. We investigated the anti-fatigue, antioxidant, and anti-inflammatory effects of eucalyptus essential oil after swimming exercise using an animal model. Male Sprague­ Dawley rats were administered eucalyptus oil (200 µL/h) daily via inhalation (15 min), and anti-fatigue effects were assessed following eucalyptus essential oil administration for 2 or 4 weeks when forced to swim until exhaustion while carrying ~5% body weight-equivalent. To assess antioxidant and anti-inflammatory effects, control and oil-treated groups were subjected to swimming, which was intensified from 90 min to 120 min daily over 4 weeks, with non-swimming groups included as controls. The 2- and 4-week-treated rats increased their swimming-to-exhaustion time by 46 s and 111 s, respectively. Additionally, lactate (LA), creatine kinase (CK), and lactate dehydrogenase (LDH) activities increased significantly in the non-treated swimming relative to levels observed in the non-swimming groups (P < 0.05); however, no significant differences in these markers were observed between the treated groups. The anti-fatigue effects were related to LA clearance and reduced LDH and CK concentrations. Moreover, compared to the corresponding levels in the non-swimmers, the non-treated swimmers showed markedly elevated levels of liver malondialdehyde (MDA), xanthine oxidase (XO), and other factors, but significantly decreased (P < 0.05) glutathione (GSH) concentrations. However, compared with that of the non-swimmer group, the treated swimming group showed no significant changes in these levels (P > 0.05), suggesting stable XO and MDA production and maintenance of GSH levels. These results suggested that eucalyptus oil aromatherapy increased rat swimming performance and antioxidant capacity and decreased oxidative damage and inflammatory reactions in tissues, indicating good anti-fatigue, antioxidant, and anti-inflammatory effects after high-intensity endurance exercise.

Kaempferol protects against propacetamol-induced acute liver injury through CYP2E1 inactivation, UGT1A1 activation, and attenuation of oxidative stress, inflammation and apoptosis in mice.

Acetaminophen (APAP) overdose can induce acute liver injury (ALI) with significant morbidity and mortality. Propacetamol is an APAP prodrug, which is clinically bioequivalent to APAP. Kaempferol, a dietary flavonoid, has antioxidant, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the protective effect of kaempferol on propacetamol-induced ALI and its underlying mechanism in mice. Kaempferol pretreatment (125 mg/kg) before propacetamol injection significantly decreased propacetamol-induced serum ALT and AST activities, and DNA fragmentation. Kaempferol administration also reduced propacetamol-induced oxidative stress by inhibiting thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine (3-NT) formation partly through downregulation of cytochrome P450 2E1 (CYP2E1) expression, upregulation of UDP glucuronosyltransferase family 1 member A1 (UGT1A1) expression, restoration of the activities of antioxidant enzymes including SOD, GPx and catalase toward normal, recovery of propacetamol-suppressed Nrf2 and GCLC expressions, and maintenance of normal glutathione level. Furthermore, kaempferol markedly attenuated APAP-induced serum TNF-α and IL-6 productions, downregulated APAP-induced phosphorylations of JNK and ERK, and decreased early hepatic apoptosis via decreasing Bax/Bcl-2 ratio and caspase 3 activation. Furthermore, administration of N-acetylcysteine (NAC) and kaempferol significantly rescued more mice than a low dose of NAC only did when a lethal dose of propacetamol injected and therapized at a delayed time point. These data suggested that kaempferol protects the liver against propacetamol-induced injury through anti-oxidative, anti-inflammatory and anti-apoptotic activities.

Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization.

Traditional serological enzyme-linked immunosorbent assay (ELISA) is routinely used to monitor pathogens during quarantine in most animal facilities to prevent possible infection. However, the ELISA platform is a single-target assay, and screening all targeted pathogens is time-consuming and laborious. In this study, to increase sensitivity and to reduce diagnosis time for high-throughput processes, multiplex PCR and DNA biochip techniques were combined to develop a multi-pathogen diagnostic method for use instead of routine ELISA. Eight primer sets were designed for multiplex PCR to detect genes from seven targeted bacterial and viral pathogens. DNA-DNA hybridization was conducted on a biochip following the multiple PCR analysis. Using this method, a total of 24 clinical samples were tested, and the result showed that not only single infection but also co-infection by multi-pathogens can be detected. In conclusion, multiplex PCR coupled with a DNA biochip is an efficient method for detecting multi-pathogens in a reaction. This platform is a useful tool for quarantine services and disease prevention in animal facilities.

Galangin ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation and cell death in mice through inhibition of ERK and NF-kappaB signaling.

Cisplatin is a chemotherapeutic agent widely used in the treatment of various cancers. However, cisplatin can induce nephrotoxicity and neurotoxicity, limiting its dosage and usage. Galangin, a natural flavonol, has been found to exhibit anti-oxidant and anti-inflammatory effects in vivo. Here, we investigated the effects of galangin on cisplatin-induced acute kidney injury (AKI) and its molecular mechanisms in mice. Galangin administration reduced the cisplatin-induced oxidative stress by decreasing renal MDA and 3-NT formations. Galangin administration also increased renal anti-oxidative enzyme activities (SOD, GPx, and CAT) and GSH levels depleted by cisplatin. Furthermore, galangin administration inactivated stress-induced Nrf2 protein and its downstream products, HO-1 and GCLC. In terms of the inflammatory response, galangin administration reduced IκBα phosphorylation, NF-κB phosphorylation and nuclear translocation, and then inhibited cisplatin-induced secretions of pro-inflammatory TNF-α, IL-1ß and IL-6. In addition, cisplatin-induced ERK and p38 phosphorylations were inhibited by galangin administration. In terms of cell death, galangin administration reduced levels of p53, pro-apoptotic Bax and activated caspase-3 to inhibit the cisplatin-induced apoptosis. Galangin administration also reduced the expression levels of RIP1 and RIP3 to inhibit cisplatin-induced RIP1/RIP3-dependent necroptosis. Therefore, galangin administration significantly ameliorates cisplatin-induced nephrotoxicity by attenuating oxidative stress, inflammation, and cell death through inhibitions of ERK and NF-κB signaling pathways. Galangin might be a potential adjuvant for clinical cisplatin therapy.

Rapid screening of roundup ready soybean in food samples by a hand-held PCR device.

Insulated isothermal PCR (iiPCR) method was recently available for rapid on-site detection of roundup ready soybean (RRS; event GTS40-3-2) in food materials and products. Performance of this method was evaluated in this study. The 100% detection endpoint for the RRS by iiPCR was found in samples containing 0.1% RRS, equivalent to the results of the reference real-time PCR (rtPCR). Analysis of nucleic acids of soybean-based processed food products indicated 95% agreement between the iiPCR and rtPCR for RRS detection. By testing soybean milk and tofu samples using simple pretreatment methods, we found that the agreements between iiPCR and rtPCR methods of the aforementioned samples were 80% and 90%, respectively. Replicated tests of all discrepant samples implied that these samples had trace amounts of RRS, suggesting that the iiPCR system is more sensitive than the rtPCR method. In conclusion, the iiPCR technology can be a useful point-of-need tool to help make a timely decision in the consumption of genetically modified organisms.

Galangin Prevents Acute Hepatorenal Toxicity in Novel Propacetamol-Induced Acetaminophen-Overdosed Mice.

Acetaminophen (APAP) overdose causes severe liver and kidney damage. APAP-induced liver injury (AILI) represents the most frequent cause of drug-induced liver failure. APAP is relatively insoluble and can only be taken orally; however, its prodrug, propacetamol, is water soluble and usually injected directly. In this study, we examined the time-dependent effects of AILI after propacetamol injection in mice. After analyses of alanine aminotransferase and aspartate aminotransferase activities and liver histopathology, we demonstrated that a novel AILI mouse model can be established by single propacetamol injection. Furthermore, we compared the protective and therapeutic effects of galangin with a known liver protective extract, silymarin, and the only clinical agent for treating APAP toxicity, N-acetylcysteine (NAC), at the same dose in the model mice. We observed that galangin and silymarin were more effective than NAC for protecting against AILI. However, only NAC greatly improved both the survival time and rate consequent to a lethal dose of propacetamol. To decipher the hepatic protective mechanism(s) of galangin, galangin pretreatment significantly decreased the hepatic oxidative stress, increased hepatic glutathione level, and decreased hepatic microsomal CYP2E1 levels induced by propacetamol injection. In addition, propacetamol injection also reproduced the probability of APAP-induced kidney injury (AIKI), appearing similar to a clinical APAP overdose. Only galangin pretreatment showed the protective effect of AIKI. Thus, we have established a novel mouse model for AILI and AIKI using a single propacetamol injection. We also demonstrated that galangin provides significant protection against AILI and AIKI in this mouse model.

Double homeobox gene, Duxbl, promotes myoblast proliferation and abolishes myoblast differentiation by blocking MyoD transactivation.

Homeobox genes encode transcription factors that regulate embryonic development programs including organogenesis, axis formation and limb development. Previously, we identified and cloned a mouse double homeobox gene, Duxbl, whose homeodomain exhibits the highest identity (67 %) to human DUX4, a candidate gene of facioscapulohumeral muscular dystrophy (FSHD). Duxbl proteins have been shown to be expressed in elongated myocytes and myotubes of trunk and limb muscles during embryogenesis. In this study, we found that Duxbl maintained low expression levels in various adult muscles. Duxbl proteins were induced to express in activated satellite cells and colocalized with MyoG, a myogenic differentiating marker. Furthermore, Duxbl proteins were not detected in quiescent satellite cells but detected in regenerated myocytes and colocalized with MyoD and MyoG following cardiotoxin-induced muscle injury. Ectopic Duxbl overexpressions in C2C12 myoblast cells promoted cell proliferation through mainly enhancing cyclin D1 and hyper-phosphorylated retinoblastoma protein but reducing p21 expression. However, Duxbl overexpression in C2C12 cells inhibited myogenic differentiation by decreasing MyoD downstream gene expressions, including M-cadherin, MyoG, p21 and cyclin D3 but not MyoD itself. Duxbl overexpressions also promoted cell proliferation but blocked MyoD-induced myogenic conversion in multipotent mesenchymal C3H10T1/2 cells. In addition, results of a luciferase reporter assay suggest that Duxbl negatively regulated MyoG promoter activity through the proximal two E boxes. In conclusion, these results indicate that Duxbl may play a crucial role in myogenesis and postnatal muscle regeneration by activating and proliferating satellite and myoblast cells.

Gcse, a novel germ-cell-specific gene, is differentially expressed during meiosis and gametogenesis.

Gametogenesis is a complex process wherein germ cells develop from primordial diploid cells into haploid gametes. To understand the mechanisms controlling gametogenesis, we identified a novel germ-cell-specific gene, Gcse. Gcse produces two major transcripts that are 1589 bp (Gcse-l) and 906 bp (Gcse-s) in length. Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR) analyses of multiple tissues reveal that Gcse-l is expressed in both adult testes and ovaries, but Gcse-s is expressed only in adult testes. During female gonad development, Gcse-l is expressed from embryonic day 13.5 to adulthood, specifically in oocytes, and maintained in ovulated and fertilized eggs. However, Gcse-s signals were detected only in ovulated oocytes and fertilized eggs but not in adult ovary. During male gonad development, strong Gcse-l signals were detected in late pachytene spermatocytes and round spermatids. However, Gcse-s transcripts exist only in round spermatids. Furthermore, the expression of GCSE-L proteins and their subcellular localizations within cells are stage specific. GCSE-L is detected in the nucleus of late pachytene spermatocytes. During meiosis, GCSE-L is translocated to acrosome regions in spermatids and maintained in the acrosome of spermatozoa. GCSE-L colocalizes with acrosin and lectin peanut agglutinin in the Golgi apparatus. However, GCSE-S proteins are expressed only in the nucleus of spermatids. From these results, we suggest that GCSE proteins play roles in meiosis and may be involved in acrosome biogenesis during spermiogenesis.

Characterization of axon formation in the embryonic stem cell-derived motoneuron.

The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.