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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)-1β, 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.

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

Chrysanthemum morifolium Ramat is traditionally used as both medicine and food in China. In this study, extracts of C. morifolium Ramat Hang Ju No. 1 (No. 1) and No. 2 (No. 2) were produced using four different solvents: 95% ethanol, ethyl acetate, n-hexane and distilled water. In total, eight types of extracts were analyzed for extraction yields and total flavonoids, polyphenols, glycans, reducing sugars, and chlorogenic acids. The antioxidant capacities and tyrosinase-inhibitory activities of these extracts were also determined. Among them, the ethanolic extract of No. 1 (No. 1A) had the highest levels of total flavonoids (16.71 mg rutin equivalent/g dry weight (DW)), polyphenols (7.07 mg gallic acid equivalent/g DW), and chlorogenic acids (6595.46 μg/g DW) and the water extract of No. 1 (No. 1D) had the highest levels of total glycans (9.24 mg/g DW), and reducing sugars (23.32 μg/g DW). In terms of antioxidant capacity, No. 1A (1.0 mg/mL) demonstrated the best 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (96.2 ± 0.4%), ferrous ion chelating ability (55.44 ± 0.03%), and reducing power (0.988 ± 0.003). No. 1D (1.0 mg/mL) showed the highest tyrosinase inhibitory activity (39.34 ± 0.03%). From these results, high levels of total flavonoids and polyphenols correlate with antioxidant capacity. Moreover, high levels of total chlorogenic acid in No. 1A and No. 1D correlate with high levels of tyrosinase inhibitory activity. Therefore, No. 1A has the potential to be used in daily health drinks, foods and skin whitening products. These results can be applied to similar flower plant extracts.

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.

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.

Previous studies identified that extracts of Talinum triangulare rich in flavonoids and phenolic acids showed antioxidative and immunomodulatory activities. In this study, the L9 orthogonal array was used to determine the optimal extraction conditions for water-extracted polysaccharides of T. triangulare (TTP) by hot reflux extraction and ultrasonic assisted extraction (UAE) methods. Results showed that while both extraction methods obtained a maximum polysaccharide yield of 3.1%, the optimal conditions for obtaining TTP was by UAE method. TTP was separated into large (LTTP) and small (STTP) molecular weights by dialysis. Since LTTP showed better effects than STTP in inducing macrophages to produce nitric oxide (NO) and indirectly inhibiting human cervical cancer HeLa cells, six different LTTP fractions were separated using anion-exchange chromatography. Contents of polysaccharides, triterpenoids, polyphenols, and proteins and molecular weights of major polysaccharide in each fraction were analyzed. The F1 fraction of LTTP, which showed the highest inducing ability of mouse RAW264.7 macrophages to secrete NO and tumor necrosis factor-α, showed the most significant indirect inhibitory effect of human colon cancer SW620 cells. These results suggest that LTTP, especially the F1 fraction, of T. triangulare may be used in health foods or Chinese medicine for its immunomodulatory potential.

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.

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 (P0.05); 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 (P0.05) glutathione (GSH) concentrations. However, compared with that of the non-swimmer group, the treated swimming group showed no significant changes in these levels (P0.05), 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.

Lipopolysaccharide (LPS) released from gram-negative bacteria stimulates immune responses in infected cells. Epigenetic modifications such as DNA methylation and protein methylation modulate LPS-induced innate immune gene expressions. Expression of the Klotho protein decreased with LPS treatment in rats. In a cellular model, information regarding the effect of LPS on Klotho expression was meager. In the present study, we demonstrated that LPS triggered global DNA and protein methylation in glomerular mesangial MES-13 cells. LPS upregulated protein expressions of enzymes central to cellular methylation reactions, especially protein arginine methyltransferase 6 (PRMT6) in MES-13 cells. Expression of the Klotho protein was diminished by LPS and was restored by 5-Aza-2'-deoxycytidine (5-Aza-2'-dc), AMI-1, and ammonium pyrrolidinedithiocarbamate (PDTC), but not adenosine aldehyde (AdOx). NF-κB was identified as a substrate for arginine methylation and interacted with PRMT6 in MES-13 cells. Inhibition of PRMT activity by AMI-1 blocked LPS-induced NF-κB nuclear translocation in MES-13 cells. Our data indicate that NF-κB negatively regulated Klotho expression with an interaction with PRMT6, which was upregulated by LPS in MES-13 cells.

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-1β 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.

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.

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.

Oral mucositis (OM) is the oral inflammation as manifestation of chemotherapy and/or radiotherapy. Cinnamomum osmophloeum (CO), of which the constituents possess anti-inflammatory activities, may have potential to alleviate OM. In this study, laboratory rats were injected with 5-Fluororacil and their oral mucosa were irritated by 18-gauge needle pouching to induce OM. Rats were randomly divided into six experimental groups: without treatment (WT), only 100 mg/mL CO leaf extract (COLE) treatment (100-only), only 5-Fluororacil treatment (5-FU), 5-FU then treated with Triamcinolone acetonide orobase (5-FU+G), 5-FU then treated with 50 mg/mL COLE, and 5-FU then treated with 100 mg/mL COLE (5-FU+100). Body weights and food and water intakes during the experimental period were recorded. Macroscopic examination, histopathological analyses, and serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels of these rats were evaluated or determined. No significant difference was found between the WT and 100-only groups. Results of macroscopic examinations, histopathological analyses, body weight changes, food and water intakes, and serum IL-1β and IL-6 levels showed significant therapeutic effects of the 5-FU+100 group compared to the 5-FU group. These finding suggest that COLE can be one of potential remedies for OM therapy through influencing proinflammatory cytokine levels.

Although Ti6Al4V alloy is commonly used as a surgical implant, the toxicity of vanadium is always a serious concern. As a stable and protective coating for Ti6Al4V, TiN deposited using a high-power impulse magnetron sputtering (HiPIMS) technique exhibits great polarization resistance in simulated body fluid and shows excellent cell viability of osteoblast-like MG63 cell. Herein, we report further study on the in vitro and in vivo biocompatibility of the HiPIMS-deposited TiN coatings with various surface modifications. For the in vitro study, the effect of O2 plasma treatment, acrylamide (AAm) graft polymerization, and bone morphogenetic protein-2 (BMP2) immobilization on the viability of osteoblast-like MG63 cell were examined using the MTT assay. For the in vivo study, the pro-inflammatory cytokines and skin pathology in mice using subcutaneous sensitivity test were conducted. In vitro investigation shows that cell viability is significantly improved for TiN having BMP2 on the surface. When BMP2 was coated on bare TiN, the MG63 cell proliferation decreases with time. However, with surface pre-treatment of TiN using O2 plasma and AAm grafting, BMP2 coated TiN exhibits increasing MG63 cell proliferation with time. Likewise, in vivo investigation shows such sample has enhanced wound healing capability, significant increase of the body weights, obvious decrease of pro-inflammatory cytokines, and improved skin section structure, as compared to the bare Ti6Al4V. The pathological result shows that the TiN treated with O2 plasma and AAm graft polymerization, and with a surface BMP2 layer gives long-term stable immobilization of BMP2 and the best in vivo biocompatibility among all the samples.

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.

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.

Aristolochic acid (AA) is a common cause of Chinese herb nephropathy. The mechanisms involved in the pathogenesis of AA nephropathy (AAN) are intricate. One well-documented effect of AA in the kidney is its pro-fibrotic activity. Nitric oxide (NO), a messenger gas generated from l-arginine, is the product of nitric oxide synthase (NOS). NO is involved in renal hemodynamics and exerts cytoprotective effects against renal injury. In the present study, the role of NO in AAN was investigated in MES-13 cells, a glomerular mesangial cell line. NO endogenously generated by the induction of inducible nitric oxide synthase (iNOS) with lipopolysaccharide (LPS)/interferon-γ (IFN-γ) significantly downregulated connective tissue growth factor (CTGF) protein expression in MES-13 cells. AA significantly suppressed LPS/IFN-γ-induced NO production and reversed CTGF expression that was downregulated by LPS/IFN-γ. AA decreased iNOS gene and protein expressions in a concentration-dependent manner. AA caused declines in LPS/IFN-γ-induced signal transducer and activator of transcription-1α (STAT-1α) phosphorylation and interferon response factor-1 (IRF-1) mRNA expression. Furthermore, AA attenuated IκB phosphorylation and reduced NF-κB translocation to the nuclear fraction. Taken together, our data indicate that AA reversed the CTGF expression inhibited by LPS/IFN-γ treatment via suppression of NO and iNOS expressions in MES-13 cells through inhibition of the JAK/STAT-1α and NF-κB signaling pathways. NO potentially exerts antifibrotic activity by down regulation of CTGF in MES-13 cells and inhibition of the iNOS gene by AA might partially account for the fibrotic effects of AA in nephropathy.

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.

Currently, no curative treatment is available for spinal muscular atrophy (SMA). Since the degeneration of spinal motor neurons in SMA is mediated by apoptosis, over-expression of an anti-apoptotic factor, Bcl-x(L), may benefit SMA. Here, we crossed a mouse model of SMA with Bcl-x(L) transgenic mice to create SMA/Bcl-x(L) mice. The Bcl-x(L) expression in the spinal neurons of SMA/Bcl-x(L) mice was nearly double that in SMA mice. SMA/Bcl-x(L) mice showed preserved motor function, normalized electrophysiological tests, diminished muscle atrophy, and less motor neuron degeneration. In addition, the life span of SMA/Bcl-x(L) mice was 1.5 times longer than that of SMA mice. Therefore, over-expression of Bcl-x(L) has a potential for amelioration of SMA, and Bcl-x(L) may be another attractive therapeutic target other than survival motor neuron (SMN) protein for use in future drug screening for SMA.

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.

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.

In this study, leaves and stems of Talinum triangulare were sequentially extracted with phosphate buffer solution to obtain PTL and PTS (phosphate buffered extracts of T. triangulare leaves and stems), with 75% ethanol to obtained ETL and ETS (ethanol extracts of T. triangulare leaves and stems), or with 90°C boiling water to obtain WTL and WTS (water extracts of T. triangulare leaves and stems). We investigated the antioxidant activities of various T. triangulare extracts, analyzed the extracts' stimulations on human mononuclear cell (MNC) growth and secretion of cytokines (interleukin-1 beta, interferon-γ, and tumor necrosis factor-α) and nitric oxide, and then assayed their subsequent inhibitions on human leukemic U937 cell growth. Results indicated that extracts of T. triangulare showed significant antioxidant activities. Among these extracts, WTS showed the highest stimulatory effect on human MNC growth. The secretion levels of interleukin-1 beta, interferon-γ, and tumor necrosis factor-α in the conditioned medium, wherein human MNC was treated with 500 μg/mL WTS for 72 hours, were 1275, 859, and 2222 pg/mL, respectively. All conditioned media obtained from human MNCs cultured with various T. triangulare extracts showed significant inhibition against U937 cell growth of over 40%. These results suggest that T. triangulare extracts may be used in health foods for their immunomodulatory potential.

The conventional translation initiator sequence for heterologous expression of metallothionein III (MTIII) in Saccharomyces cerevisiae was replaced by a yeast consensus initiator region (CAAAAUG). Saccharomyces cerevisiae was transformed with a vector carrying the MTIII gene and the modified initiator sequence had a much higher metal tolerance, Cd accumulation and MTIII production than that with the original initiator sequence, although the level of gene transcripts was similar in both systems. This modification markedly improves MTIII production and may also be used for production of other genes.

Growth inhibitory factor (GIF), a member of the metallothionein (MT) family, is also known as MTIII. This protein distinguishes itself from other MT isoforms by exerting an inhibitory effect on cortical neuron growth instead of metal ion buffering. In this work, we cloned MTIII genes from a porcine brain cDNA library. Two species of clones were isolated that vary with respect to one nt in the coding sequence. This discrepancy results in the translation of two MTIII primary structures having a different amino acid at residue 46. Herein, both MTIII cDNAs were constructed into an expression vector and transformed into yeast cells, respectively. The yeast carrying either MTIII gene displayed a similar metal tolerance when cultured in a medium containing metal. The resistance to metal toxicity was attributed to the expression of MTIII gene which was confirmed by RNA and protein analyses. The characteristics of the protein stability, metal binding affinity and ultraviolet absorption spectrum of the yeast produced MTIII are also compared with those of MTII. The comparison reveals that both MTs have similar physical characteristics. Moreover, circular dichroism spectrum of Cd saturated MTIII was analyzed as well. Typical Cys-Cd bands for MTII appear in the spectrum, indicating similar metal-thiol interactions for MTIII to those for other MT isoforms.

The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose- and time-dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S-cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells.

It has been reported that the anti-inflammatory activity of 3-hydroxy-3-methyl-glutary coenzyme A (HMG-CoA) reductase inhibitors (statins) is independent of their hypocholesterolemic effect. Previous studies indicated that induction of heme oxygenase-1 (HO-1) exerts a cytoprotective activity in several inflammatory diseases. Here, the possibility that HO-1 is involved in the anti-inflammatory action of simvastatin, using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages as a model system has been specifically addressed. Our results demonstrated that in the presence of LPS, simvastatin significantly increased HO-1 expression and activity in a dose-dependent manner compared to that of LPS-stimulated alone macrophages. Moreover, simvastatin significantly inhibited LPS-induced inducible nitric oxide synthase (NOS) expression, and formation of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), nitrite and free radicals, but enhanced interleukin-10 (IL-10) production. Similarly, the IκB-α degradation and nuclear transcription factor-κB translocation and activation caused by LPS were significantly suppressed by simvastatin. However, these anti-inflammatory activities of simvastatin were markedly reversed by addition of a HO-1 inhibitor zinc protoporphyrin (ZnPP). Accordingly, the present results indicate that the anti-inflammatory activity of simvastatin could, at least in part, be regulated by induction of HO-1-mediated processes.

We identified and cloned a mouse double homeobox gene (Duxbl), which encodes two homeodomains. Duxbl gene, a tandem triplicate produces two major transcripts, Duxbl and Duxbl-s. The amino acid sequences of Duxbl homeodomains are most similar to those of human DUX4 protein, associated with facioscapulohumeral muscular dystrophy. In adult tissues, Duxbl is predominantly expressed in female reproductive organs and eyes, and slightly expressed in brain and testes. During gonad development, Duxbl is expressed from embryonic to adult stages and specifically expressed in oocytes and spermatogonia. During embryonic development, Duxbl is transcribed in limbs and tail. However, Duxbl proteins were only detected in trunk and limb muscles and in elongated myocytes and myotubes. In C2C12 muscle cell line, Duxbl expression pattern is similar to differentiated marker gene, Myogenin, increased in expression from 2 days onward in differentiating medium. We suggest that Duxbl proteins play regulatory roles during myogenesis and reproductive developments. Developmental Dynamics 239:927–940, 2010. © 2010 Wiley-Liss, Inc.

The homeobox gene families play important roles in the transcriptional regulation of gene expression prior to and during embryo development. To identify novel homeobox genes expressed in early embryonic development, we conducted a degenerated oligonucleotide polymerase chain reaction (PCR) to screen a mouse embryonic stem (ES) cell cDNA library. A novel homeobox-containing gene, Eso-1, which is preferentially expressed in ES cells and ovaries, was identified. The full-length Eso-1 cDNA was found to be 1,710 bp with a predicted homeodomain that has no significant homology to previously reported home- odomain proteins. Eso-1 was mapped to chromosome 14A3. Reverse transcription-polymerase chain reac- tion (RT-PCR) analyses showed that Eso-1 was expressed through oogenesis and continuing to be expressed through to the blastocyst stage. De novo expression of Eso-1 started at 13.5 days postcoitum in the ovaries, which coincides with the initiation of oogenesis. Northern blot analyses demonstrated that Eso-1 is preferentially expressed in both ovaries and ES cells as a 1.7-kb transcript. Results from whole mount in situ hybridization revealed that Eso-1 in oocytes showed increased expression from primordial to antral follicles. The 3 0 -untranslated region of Eso-1 transcripts contained cytoplasmic polyadenylation sequences while the length of poly (A) tails changed during oocyte maturation, indicating that Eso-1 expression is con- trolled by time-dependent translational activation. We suggest that the novel homeodomain protein, Eso-1, plays a role during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 73: 825-833, 2006. 2006 Wiley-Liss, Inc.

Spinal muscular atrophy (SMA) is the most common genetic motoneuron degenerative disorder, but the mechanism(s) of motoneuron death is unclear. Previously, a direct interaction between tumor-suppressive TP53 protein and the SMA determinant gene product, survival motor neuron protein, was identified and therefore it has been suggested that a mechanism of TP53-dependent apoptosis plays an important role in motoneuron degeneration in SMA. We used our SMA model mice, generated by a combination of knockout and transgenic techniques, to decipher the role of TP53 protein in the motoneuron degeneration in SMA. We detected a significant increase of Trp53 expression in the spinal cord of SMA-like mice compared to their normal littermates. After crossing SMA-like mice with Trp53 knockout mice, the progeny Trp53-deficient SMA-like mice did not show milder disease severity or longer lifespan compared to SMA littermates with wild-type Trp53 genes. Our studies provide in vivo evidence indicating that Trp53-dependent apoptosis does not play a crucial role in motoneuron degeneration in SMA-like mice. European Journal of Human Genetics (2006) 14, 372-375. doi:10.1038/sj.ejhg.5201556; published online 4 January 2006.

In order to identify novel homeobox-containing genes involved in early embryonic development, we conducted a degenerate oligonucleotide polymerase chain reaction (PCR) screening of a murine embryonic stem (ES) cell cDNA library. ENK (early embryo specific expression NK family) was one of several genes isolated that was found to exhibit early embryo stage-specific expression. The full-length ENK cDNA was cloned and its genomic organization was characterized. Murine ENK spans 7.1 kb, encodes four exons and maps to mouse chromosome 6F2. Reverse transcriptase-PCR and Northern blot analyses show that ENK is preferentially expressed in pre-implantation mouse embryos and a higher level in blastocysts. Whole-mount in situ hybridization analysis further demonstrates that ENK mRNA is present predominantly in the inner cell mass of blastocysts. The expression of ENK is markedly higher in undifferentiated ES cells than in retinoic acid differentiated ES cells and embryonic bodies. ENK expression slightly decreased in early primitive ectoderm-like (EPL) cells and was absent after the 9.5-day embryo stages. ENK is one of the few homeobox-encoding genes preferentially expressed in ES cells during mammalian embryogenesis.

Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene. Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs. In contrast, P. pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form. Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 microM) resulted in significant improvements in expression of rPLF from P. pastoris. Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L). P. pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column. The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF.

Metallothionein ( MT ) cDNAs were cloned and sequenced from two genera of ducks, Muscovy ( Cairina muschata ) and Tsai ya ( Anas platyrhynchos ). The two cDNAs show an extremely high sequence homology and contain an open reading frame encoding 63 amino acids. MT mRNA expressions were studied after metal induction using the cloned cDNA as a probe. Cadmium and copper induced MT gene efficiently, whereas zinc showed a markedly less effect. In addition, the MT mRNA accumulations in various developmental stages were also investigated. The result reveals a different pattern of expression from that of mammals. The discrepancy in MT gene between Tsai ya and Muscovy was further explored by examining genomic DNA structures. The duck MT showed three exons and two introns. The most significant variation of the genes occurs at intron II in which Tsai ya MT has 24 bases more than Muscovy MT . Moreover, MT expressions in the hybrids of Muscovy and Tsai ya were investigated using a reverse transcriptase-polymerase chain reaction. Those results demonstrated that both parental MT genes are expressed in the hybrids after metal induction.

Porcine metallothionein III (MTIII) was isolated from brain tissue by the combination of gel filtration and anion-exchange chromatographies. The identity of MTIII was confirmed by comparing the chromatographic characteristics to other MT isoforms isolated from porcine liver. Porcine MTIII has a low molecular weight and a pl of 4.1. This protein contains both zinc and copper, and the zinc can be replaced by cadmium. Using reverse transcriptase-polymerase chain reaction, a 0.2 kb DNA fragment can be amplified from the porcine brain mRNA. This DNA fragment was demonstrated to contain MTIII coding region after cloning and sequencing. The revealed DNA sequence can be translated into 68 amino acids and shows a common structural characteristic of MTIII from other species. Northern blot analysis indicated that MTIII inRNA is expressed in every specified region of the porcine brain examined here.