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A gene encoding an archaeal transcription regulator (TON_1525) homologous to the Tfx family DNA-binding protein has been identified to affect gene expression of carbon monoxide metabolism in the hyperthermophilic archaeon Thermococcus onnurineus NA1. To broadly understand gene regulation by the TON_1525 gene, a gene deletion mutant (Δ1525) was constructed and transcriptome changes were investigated. A total of 351 genes were found to be differentially expressed in the Δ1525 mutant. The expression levels of the hydrogenase genes were significantly changed; mbh (membrane-bound hydrogenase) and mch (membrane-bound carbon monoxide-dependent hydrogenase) genes were increased, and mfh 2 (membrane-bound formate-dependent hydrogenase) and soluble hydrogenase genes were decreased. The Δ1525 mutant was cultured in a medium supplemented with maltodextrin or carbon monoxide to investigate whether changes in gene expression lead to physiological changes. The maximum H 2 production rates of the Δ1525 mutant were significantly enhanced on both substrates compared to the wild-type strain. These results indicate that the TON_1525 gene can affect the expression of various genes including mbh and mch , consequently regulating the metabolism of T. onnurineus NA1. This study expands our understanding of the functional role of the Tfx family DNA-binding proteins. • Functional studies on the role of the Tfx family DNA-binding protein are limited. • Deletion of the TON_1525 gene caused expression changes in various genes. • The Δ1525 mutant showed enhanced H 2 production by upregulation of H 2 evolving hydrogenase genes. [ABSTRACT FROM AUTHOR]

Patients with dementia are diagnosed with deficiency patterns and interior patterns in traditional Chinese medicine due to decreased physical strength, mental atrophy including cognitive function, and decreased motor function in the gastrointestinal tract. Since "greater yin symptom" in Shanghanlun has been interpreted as interior, deficiency, and cold pattern in traditional Chinese medicine, it is necessary to determine whether Geijigadaehwang-tang (GDT) has therapeutic effects on neurodegenerative diseases and the underlying mechanism if it has such effects. Aims of the study: Trimethyltin (TMT), a neurotoxic organotin compound, has been used to induce several neurodegenerative diseases, including epilepsy and Alzheimer's disease. This study aimed to evaluate the therapeutic efficacy of GDT for TMT-induced hippocampal neurodegeneration and seizures and to determine the mechanisms involved at the molecular level. The main components of GDT were analyzed using ultra-performance liquid chromatography. TMT was used to induce neurotoxicity in microglial BV-2 cells and C57BL6 mice. GDT was administered at various doses to determine its neuroprotective and seizure inhibition effects. The inhibitory effects of GDT on TMT-induced apoptosis, inflammatory pathways, and oxidative stress pathways were determined in the mouse hippocampal tissues. GDT contained emodin, chrysophanol, albiflorin, paeoniflorin, 6-gingerol, and liquiritin apioside. In microglial BV-2 cells treated with TMT, GDT showed dose-dependent neuroprotective effects. Oral administration of GDT five times for 2.5 days before and after TMT injection inhibited seizures at doses of 180 and 540 mg/kg and inhibited neuronal death in the hippocampus. In hippocampal tissues extracted from mice, GDT inhibited the protein expression of ionized calcium binding adaptor molecule 1, glial fibrillary acidic protein, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3, and phosphorylated nuclear factor (NF)-κB/total-NFκB ratio. Additionally, GDT inhibited the messenger RNA levels of tumor necrosis factor-α, inducible nitric oxide synthase, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, interleukin-1β, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1. This study's results imply that GDT might have neuroprotective potential in neurodegenerative diseases through neuronal death inhibition and anti-inflammatory and antioxidant mechanisms. [Display omitted] [ABSTRACT FROM AUTHOR]

Rapid assembly of DNA damage response (DDR) proteins at nuclear “repair” foci is a hallmark response of ionizing radiation (IR)-treated cells. The ubiquitin E3 ligases RNF8 and RNF168 are critical for foci formation, and here we aim to determine their dynamic mobility and abundance at individual foci in living cells. To this end, YFP-tagged RNF8 and RNF168 were expressed at physiological levels in MCF-7 cells, then analyzed by fluorescence recovery after photobleaching (FRAP) assays, nuclear retention measurement, and virus-like particles (VLPs)-based quantification. The results showed that RNF8 and RNF168 were both highly dynamic at IR-induced foci. Intriguingly, RNF8 displayed remarkably faster in vivo association/dissociation rates than RNF168, and RNF8-positive IR-foci were less resistant to detergent extraction. In addition, copy number assay revealed that RNF168 was two-fold more abundant than RNF8 at foci. Collectively, we show for the first time that RNF8 moves on-and-off nuclear DNA repair foci more than six-fold as quickly as RNF168. The faster kinetics of RNF8 recruitment explains why RNF8 is generally observed at DNA-breaks prior to RNF168. Moreover, our finding that RNF8 is less abundant than RNF168 identifies RNF8 as a rate-limiting determinant of focal repair complex assembly. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) generally pair with the 3′UTRs of their target mRNAs to repress gene expression. It has reported that miRNA targets (TGs) are longer and evolve more slowly than non-targets (NTGs). We confirmed the observation and also found novel structural and expressional characteristics of TGs. The length difference between TGs and NTGs was greatest for the 3′UTRs, although a difference was also observed for CDSs and introns. Widely expressed genes were shorter for both TGs and NTGs; however, TGs were significantly longer than NTGs in all ranges of expression. TGs were more likely than NTGs to be widely expressed, which might explain why TGs evolve more slowly than NTGs. Finally, we found that TG mRNAs have faster decay rates. In addition, the decay rate of a TG mRNA transcript was found to be positively correlated with the number or density of target sites located in that TG''s mRNA transcript. [Copyright &y& Elsevier]

Abstract: Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we report that multimerized siRNA conjugate composed of two different siRNA sequences in the same backbone shows more efficient inhibition of the two corresponding target genes at one time than physically mixed multimerized siRNA conjugates. Two model siRNAs against VEGF and GFP gene were chemically crosslinked via cleavable and noncleavable linkages for the preparation of dual gene targeted multimeric siRNA conjugates (DGT multi-siRNA). Cleavable DGT multi-siRNA with reducible disulfide linkages exhibited significantly higher gene silencing efficiencies at mRNA and protein expression levels than noncleavable DGT multi-siRNA, the physical mixture of naked siRNA, and that of single gene targeted multimeric siRNA (SGT multi-siRNA) with eliciting negligible immune response. DGT multi-siRNAs against two therapeutic siRNAs, anti-survivin and anti-bcl-2 targeted siRNA, also showed greatly enhanced apoptotic effect. This approach for concurrent suppression of combinatorial therapeutic target genes using cleavable multimeric siRNA structure can be potentially used for improved therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding a serine protease inhibitor of Clonorchis sinensis (CsSERPIN) was identified and characterized. CsSERPIN contained an open reading frame of 1158bp that encoded 385 amino acid residues. Sequence analysis of the primary structure of CsSERPIN revealed that it had essential structural motifs including a reactive central loop (RCL), which well conserved in the serine protease inhibitor (serpin) superfamily. CsSERPIN was classified as a member of the ovalbumin-type serpin family on the basis of phylogenetic analysis and the absence of a classical N-terminal signal peptide. Recombinant CsSERPIN showed an inhibitory effect on chymotrypsin in a dose-dependent manner, but did not effectively inhibit trypsin, thrombin, elastases or cathepsin G. Optimal pH values of CsSERPIN were between 7.0 and 9.0, as evidenced by the rapid loss of inhibitory activity under acidic conditions. CsSERPIN was expressed at various developmental stages of the parasite, from eggs to adult worms, but its expression level was higher in eggs and adult worms than in metacercariae and juvenile worms. CsSERPIN was identified in the soluble extract of the parasite, but not in the excretory and secretory products (ESP) or insoluble extract of the parasite. Immunolocalization analysis of CsSERPIN showed that it mainly localized to the eggs and vitelline glands of the adult worm. These results suggest that intracellular CsSERPIN may be possibly involved in maintaining the physiology of eggs as well as in egg production of C. sinensis by regulating endogenous serine proteases. [ABSTRACT FROM AUTHOR]

Summary: Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. Here, we identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors. Independent evaluation confirmed the association of a prognostic gene microfibril-associated glycoprotein 2 (MAGP2) with poor prognosis, whereas in vitro mechanistic analyses demonstrated its ability to prolong tumor cell survival and stimulate endothelial cell motility and survival via the αVβ3 integrin receptor. Increased MAGP2 expression correlated with microvessel density suggesting a proangiogenic role in vivo. Thus, MAGP2 may serve as a survival-associated target. [Copyright &y& Elsevier]

Abstract: Proline-rich membrane anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G4) that anchors onto the plasma membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G4 AChE when that was over expressed together with AChET subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G4 AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles. [Copyright &y& Elsevier]

Abstract: A good understanding of telomeres and telomerase biology is crucial for unraveling mechanisms related to aging and cancer. However, in vivo vertebrate studies of telomere biogenesis and telomerase function have been limited by the development of appropriate animal model systems. The present study aims to demonstrate evolutionary conservation of telomerase in vertebrate species, supporting the potential application of fish as vertebrate model for studying telomeres and telomerase function. Comparison of genomic and protein information among vertebrate TERTs (TElomerase Reverse Transcriptase), the Japanese medaka Oryzias latipes shares the highest similarity to that of the human than the other small size fish species studied (including pufferfish and zebrafish). The ubiquitous expression of TERT mRNA, the high constitutive level of telomerase activity, and the humanized telomere lengths further substantiate that Japanese medaka is an ideal vertebrate model for the study of telomere and telomerase-related mechanisms in vivo. Moreover, medaka exhibits fast, invariable growth and is able to provide a variety of useful developmental and reproductive endpoints for lifelong and multi-generational experiments. Our earlier and present findings support the use of medaka for studying organismal aging, tissue regeneration and carcinogenesis. [Copyright &y& Elsevier]

Cannabidiol, a non-psychoactive phytocannabinoid, has antioxidant and anti-inflammatory activity in keratinocytes. However, the signaling pathway through which cannabidiol exerts its effect on keratinocytes or whether it can modulate keratinocyte differentiation has not been fully elucidated yet. We investigated whether cannabidiol modulates epidermal differentiation and scavenges reactive oxygen species through the aryl hydrocarbon receptor (AhR) in keratinocytes and epidermal equivalents. We investigated the cannabidiol-induced activation of AhR using AhR luciferase reporter assay, qRT-PCR, western blot, and immunofluorescence assays. We also analyzed whether keratinocyte differentiation and antioxidant activity are regulated by cannabidiol-induced AhR activation. In both keratinocytes and epidermal equivalents, cannabidiol increased both the mRNA and protein expression of filaggrin, involucrin, NRF2, and NQO1 and the mRNA expression of the AhR target genes, including CYP1A1 and aryl hydrocarbon receptor repressor. Additionally, cannabidiol showed antioxidant activity that was attenuated by AhR knockdown or co-administration with an AhR antagonist. Moreover, cannabidiol increased the ratio of OVOL1/OVOL2 mRNA expression, which is a downstream regulator of AhR that mediates epidermal differentiation. In addition to increased expression of barrier-related proteins, cannabidiol-treated epidermal equivalent showed a more prominent granular layer than the control epidermis. The increased granular layer by cannabidiol was suppressed by the AhR antagonist. Cannabidiol can be a modulator of the AhR-OVOL1-filaggrin axis and AhR-NRF2-NQO1 signaling, thus indicating a potential use of cannabidiol in skin barrier enhancement and reducing oxidative stress. • CBD regulates AhR in keratinocytes and epidermal equivalents. • CBD induces epidermal differentiation by modulating the AhR-OVOL1 axis. • AhR orchestrates ROS scavenging through NRF2 regulation. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Lonicera japonica Thunberg is a traditional herbal medicine widely used in East Asia as an anti-bacterial, anti-inflammatory, and antiviral agent. This study was designed to investigate the effects of HS-23, ethanol extract of the dried flower buds of Lonicera japonica, in experimental models of sepsis and elucidate the mechanisms of action of HS-23. Materials and methods Male ICR mice were intravenously administered HS-23 (20 and 40 mg/kg) for 0 (immediately) and 24 h after cecal ligation and puncture (CLP) for survival tests, and HS-23 (40 mg/kg) immediately after CLP for biochemical assays. Results HS-23 improved sepsis-induced mortality, enhanced bacterial clearance, and attenuated multiple organ failure. The mechanisms of action of HS-23 included attenuation of increased toll-like receptor (TLR)4 protein and mRNA expression. HS-23 suppressed sepsis-induced increases in protein expression of myeloid differentiation primary response protein 88, p38 and c-Jun N-terminal kinase in both liver and lung, as well as TIR-domain-containing adapter-inducing interferon-β and interferon regulatory transcription factor 3 protein expression in liver. Conclusion The results of this study revealed that HS-23 attenuated sepsis through suppression of TLR signaling pathways. Therefore, our findings suggest that HS-23 might be useful as a potential therapeutic agent for treatment of sepsis. [ABSTRACT FROM AUTHOR]

Gekko gecko is used as a traditional medicine for various diseases including respiratory disorders in northeast Asian countries, mainly Korea, Japan, and China. Allergic asthma is a chronic respiratory disease caused by an inappropriate immune response. Due to the recent spread of coronavirus disease 2019, interest in the treatment of pulmonary disorders has rapidly increased. In this study, we investigated the anti-asthmatic effects of G. gecko extract (GGE) using an established mouse model of ovalbumin-induced asthma. To evaluate the anti-asthmatic effects of GGE, we evaluated histological changes and the responses of inflammatory mediators related to allergic airway inflammation. Furthermore, we investigated the regulatory effects of GGE on type 2 helper T (Th2) cell activation. Administration of GGE attenuated asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and expression of Th2 cytokines. Furthermore, GGE treatment reduced Th2 cell activation and differentiation. These results indicate that GGE alleviates allergic airway inflammation by regulating Th2 cell activation and differentiation. [Display omitted] • Gekko gecko is traditionally used as therapeutic agent to treat pulmonary diseases in Asian countries. • Gekko gecko extracts reduced inflammatory cell infiltration and goblet cell hyperplasia in airway. • Gekko gecko extracts decreased allergic inflammation by regulating Th2 cell activation and polarization. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Dual antiplatelet therapy (DAPT) with aspirin (ASA) and clopidogrel (CLP) has been consistently shown clinical effectiveness in patients with coronary artery disease. According to the literature, four traditional Chinese medicine (TCM) herbs effective for prevention cardiovascular diseases, namely Radix Salvia Miltiorrhiza (Red sage root, Danshen), Radix Pueraria Lobata (Kudzu root, Gegen), Radix Angelica Sinensis (Angelica root, Danggui), and Rhizoma Ligusticum chuanxiong (Szehuan lovage rhizome, Chuanxiong), are of high potential to be co-administered during DAPT. Since all these herbs are blood vitalizing medicines and can promote blood circulation and eliminate blood stasis, it was hypothesized that they may potentially alter the clinical outcomes of DAPT with clopidogrel and aspirin. Aim of study The current study is proposed aiming to preliminarily evaluate the impact of these four commonly used Chinese medicinal herbs on the pharmacokinetics and pharmacodynamics of the combination therapy with clopidogrel and aspirin and its relevant outcomes and mechanisms. Materials and methods In order to mimic the standard dosing regimen for DAPT in human, various Sprague-Dawley rats treatment groups were received a bolus oral dose of DAPT on day 1 followed by DAPT for consecutive 13 days in absence and presence of orally co-administered four TCM herbs (Danshen, Gegen, Danggui and Chuanxiong) at their low and high doses. On day 14, serial blood samples were collected after dosing to obtain the plasma concentrations of ASA, CLP and their corresponding metabolites by LC/MS/MS. At the end of last blood sampling point of each rat, about 4.5 ml of whole blood were collected to estimate the prothrombin time from each treatment groups. After all the blood sampling, the rats were sacrificed followed by collecting their livers for evaluations of enzyme activities and expressions in the related liver microsome preparations and stomach tissues for evaluations of their potential ulcer index. In addition, gene expression and protein levels of related biomarkers (COX-1, COX-2, P2Y12) in rat livers were measured by RT-PCR and Western blot, respectively, and compared among different treatment groups. Results Co-administration of Gegen and Danggui significantly altered the pharmacokinetics of ASA and CLP in DAPT with increased systemic exposure of ASA and CLP respectively. Although minimal impact on aspirin esterase activity for all co-administered herbs, significant inhibition on rCyp2c11 and carboxylesterase activities were observed for DAPT with Danshen, Gegen and Danggui co-treatment. In addition, significantly longer PT were found in all DAPT treatment groups. However, a trend of decrease in PT of DAPT in presence of Gegen, Danggui and Chuanxiong was noticed. Nevertheless, all the treatments did not cause detectable changes in COX and P2Y12 mRNA and protein expressions. Conclusion Among the four studied TCMs, it was demonstrated that co-administration of Gegen and Danggui could lead to altered pharmacokinetics of DAPT with significant inhibition on rCyp2c11 and carboxylesterase activities. Although Gegen, Danggui and Chuanxiong might potentially offset the anticoagulant activity of DAPT, the overall pharmacodynamics outcome was not considered to be harmful due to lack of risk in bleeding, which warrant further verification for its clinical impact. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Background Retinal degeneration is a condition ensued by various ocular disorders such as artery occlusion, diabetic retinopathy, retrolental fibroplasia and retinitis pigmentosa which cause abnormal loss of photoreceptor cells and lead to eventual vision impairment. No efficient treatment has yet been found, however, the use of stem cell therapy such as bone marrow and embryonic stem cells has opened a new treatment modality for retinal degenerative diseases. The major goal of this study is to analyze the potential of endothelial progenitor cells derived from bone marrow to differentiate into retinal neural cells for regenerative medicine purposes. Methods In this study, endothelial progenitor cells were induced in-vitro with photoreceptor growth factor (taurine) for 21 days. Subsequently, the morphology and gene expression of CRX and RHO of the photoreceptors-induced EPCs were examined through immunostaining assay. Findings The results indicated that the induced endothelial progenitor cells demonstrated positive gene expression of CRX and RHO. Our findings suggested that EPC cells may have a high advantage in cell replacement therapy for treating eye disease, in addition to other neural diseases, and may be a suitable cell source in regenerative medicine for eye disorders. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is a carcinogenic human liver fluke. Its infection promotes persistent oxidative stress and chronic inflammation environments in the bile duct and surrounding liver tissues owing to direct contact with worms and their excretory–secretory products (ESPs), provoking epithelial hyperplasia, periductal fibrosis, and cholangiocarcinogenesis. We examined the reciprocal regulation of two ESP-induced redox-active proteins, NF-κB and peroxiredoxin 6 (Prdx6), during C. sinensis infection. Prdx6 overexpression suppressed intracellular free-radical generation by inhibiting NADPH oxidase2 and inducible nitric oxide synthase activation in the ESP-treated cholangiocarcinoma cells, substantially attenuating NF-κB-mediated inflammation. NF-κB overexpression decreased Prdx6 transcription levels by binding to two κB sites within the promoter. This transcriptional repression was compensated for by other ESP-induced redox-active transcription factors, including erythroid 2-related factor 2 (Nrf2), hypoxia inducible factor 1α (HIF1α), and CCAAT/enhancer-binding protein β (C/EBPβ). Distribution of immunoreactive Prdx6 and NF-κB was distinct in the early stages of infection in mouse livers but shared concomitant localization in the later stages. The intensity and extent of their immunoreactive staining in infected mouse livers are proportional to lesion severity and infection duration. The constitutive elevations of Prdx6 and NF-κB during C. sinensis infection may be associated with more severe persistent hepatobiliary abnormalities mediated by clonorchiasis. [ABSTRACT FROM AUTHOR]

Expression of receptor for advanced glycation end-products (RAGE) is suggested to play a crucial role in mediating cardiac ischemia/reperfusion (IR) injury, and the blockade of RAGE signaling has been considered as a potential therapeutic strategy for the treatment of IR-induced cardiac damage. In this study, we primarily investigated the effects of RAGE suppression particularly on IR-induced ventricular arrhythmia. To inhibit the IR-induced upregulation of RAGE, siRNA targeting RAGE (siRAGE) was delivered to myocardium by using deoxycholic acid-modified polyethylenimine (PEI-DA) as a non-viral gene carrier. The resultant PEI-DA/siRAGE nanocomplexes successfully silenced the expression of RAGE and attenuated the inflammation and apoptosis in the ischemic-reperfused myocardium. According to our results, the electrophysiological properties (e.g., action potential propagation, action potential duration, and conduction velocity), disrupted by IR injury, were restored to normal level and the induction of ventricular tachycardia was abolished by RAGE silencing. We further found that RAGE suppression led to the activation of Wnt signaling, followed by the expression of gap junction protein, connexin43. Thus it could be concluded that successful siRAGE delivery is protective against IR-induced ventricular arrhythmia. [ABSTRACT FROM AUTHOR]

Physical exercise induces gene expression changes that trigger glucose metabolism pathways in organisms. In the present study, we monitored the expression levels of LDHA (lactate dehydrogenase) and GYS1 (glycogen synthase 1) in the blood, to confirm the roles of these genes in exercise physiology. LDHA and GYS1 are related to glucose metabolism and fatigue recovery, and these processes could elicit economically important traits in racehorses. We collected blood samples from three retired thoroughbred racehorses, pre-exercise and immediately after 30min of exercise. We extracted total RNA and small RNA (≤200 nucleotide-long) from the blood, and assessed the expression levels of LDHA, GYS1, and microRNAs (miRNAs), by using qRT-PCR. We showed that LDHA and GYS1 were down-regulated, whereas eca-miR-33a and miR-17 were up-regulated, after exercise. We used sequences from the 3' UTR of LDHA and GYS1, containing eca-miR-33a and miR-17 binding sites, to observe the down-regulation activity of each gene expression. We observed that the two miRNAs, namely, eca-miR-33a and miR-17, inhibited LDHA and GYS1 expression via binding to the 3' UTR sequences of each gene. Our results indicate that eca-miR-33a and miR-17 play important roles in the glucose metabolism pathway. In addition, our findings provide a basis for further investigation of the exercise metabolism of racehorses. [ABSTRACT FROM AUTHOR]

Highlights: [•] Photosynthetic responses of M. aeruginosa under UV-C stress was investigated. [•] Gene suppression was the primary response to UV-C, but rapidly repaired. [•] Immediate photoinhibition, F V/F M reduction, after 50–200mJcm−2 UV-C was observed. [•] Insufficient recovery processes, elevations in D1, qP and NPQ, were detected. [•] Both light-harvesting and photochemical interruptions result in growth suppression. [ABSTRACT FROM AUTHOR]

Abstract: In the mitochondrial matrix, the OXCT1 gene catalyzes the reversible transfer of coenzyme A from succinyl-CoA to acetoacetate in a reaction related to energy production from ketone bodies. Here, horse OXCT1 gene containing coenzyme A transferase domain was identified in the transcriptome analysis of cDNAs derived from skeletal muscles. Horse OXCT1 gene consisted of 1741 nucleotide sequences with an open reading frame of 1560 nucleotides encoding a protein of 520 putative amino acid residues. The number of non-synonymous substitutions was lower than the number of synonymous substitutions in the OXCT1 genes of other species, indicating that purifying selection occurred in the OXCT1 genes during evolutionary radiation. Quantitative real-time RT-RCR analysis showed a dominant expression pattern of horse OXCT1 gene in the cerebrum, heart, and skeletal muscle. Different expression levels of horse OXCT1 transcripts between before- and after-exercise samples were also measured in the skeletal muscles of six horses. These data could be of great use for further investigation of the relationship between energy products and horse OXCT1 gene. [Copyright &y& Elsevier]

Abstract: Natural Killer cells are known to play a major role in the innate immune response against viral infections and tumor cells. Several viruses, such as CMV, EBV and HIV-1, have acquired strategies to escape elimination by NK cells. In this study, we observed that EBV infection increased expression of IDO on B cells. To evaluate the function of IDO associated with EBV infection, we investigated whether EBV-induced IDO could modulate expression of NK cell-activation receptor, NKG2D. When NK cells were co-incubated with EBV transformed B cells, surface expression of NKG2D was significantly reduced in NK cells. Incubation with L-kynurenine, an IDO metabolite, down-modulated NKG2D expression in NK cells in a dose- and time-dependent manner. Incubation with the JNK inhibitor SP600125 also inhibited NKG2D expression in NK cells. In addition, we observed that the effect of L-kynurenine was blocked by JNK agonist, anisomycin, suggesting the involvement of the JNK pathway in the signal transduction of L-kynurenine-reduced NKG2D expression. Furthermore, IL-18 significantly reduced L-kynurenine-induced down-regulation of NKG2D expression in NK cells. Taken together, these data indicate that down-regulation of NKG2D by EBV-induced IDO metabolite provides a potential mechanism by which EBV escapes NKG2D-mediated attack by immune cells. [Copyright &y& Elsevier]

Abstract: Lymphocyte activation gene-3 (LAG-3; CD223) is a co-receptor that may function as a down-modulator of T cell activity via an interaction with MHC class II molecules. In this study, we cloned and sequenced the complete cDNA sequence of pig Lag3. Pig Lag3 cDNA contains an open reading frame (1524bp) encoding 507 amino acids. The putative amino acid identity of pig LAG-3 with those of the human, rat, and mouse is 78%, 66%, and 67%, respectively. Pig Lag3 mRNA transcript was mainly expressed in lymphoid tissues, and was inducible upon stimulation of phytohemagglutinin (PHA). Next, we developed mouse antiserum against the extracellular domain (D1 and D2 domains) of pig LAG-3. Pig LAG-3 is detected as an approximately 67kDa of a single species by Western blot analysis. Moreover, flow cytometry analysis confirmed that LAG-3 on pig T cells (CD3+ cells) is inducible upon PHA stimulation. These results indicated that the structure and expression pattern of pig LAG-3 are conserved among mammalian species. [Copyright &y& Elsevier]

Abstract: Mechanical loading such as interstitial fluid shear stress and tensile strain stimulates bone cells, which respond by changing bone mass and structure to maintain optimal skeletal architecture. Bone cells also adapt to bone implants and altered mechanical loading. Osseous integration between host bone and implants is a prerequisite for the stability of implants. Fluctuating fluid pressure and interfacial strains occur between bone cells and implants due to mechanical loading during walking and other daily activities. In this study, we examined the signaling mechanism by which mechanical stimulation activates a novel transcription factor in human and mouse bone cells. Nuclear factor of activated T cells (NFAT) is one of the transcription factors that act downstream of the Ca++/calcineurin (Ca++/Cn) network: a well-known pathway of inflammation. In this study, we hypothesized that NFAT2 is activated in response to mechanical stimulation and mediates Cox2 expression. Fluid shear stress and tensile strain results in nuclear translocation of NFAT in cells of the osteoblastic lineage. A peptide inhibitor of the Cn/NFAT axis was found to block the mechanical stimulation-mediated Cox2 induction. Further, chromatin immunoprecipitation assay shows direct interaction between NFAT2 and the human Cox2 promoter region. Additionally, CnAβ knockout calvarial bone cells were found to be less sensitive than control bone cells to mechanical stimulation. Our study provides new evidence for a novel role for NFAT in bone mechanotransduction in the context of cytokine gene induction in bone cells. [Copyright &y& Elsevier]

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2'-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease. [ABSTRACT FROM AUTHOR]

BACKGROUND: Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies. [ABSTRACT FROM AUTHOR]

Aberrant retinoid signaling in human cancers is extending from the nucleus to the cytoplasm. Recently, we have demonstrated frequent epigenetic inactivation of a retinoic acid receptor (RAR), RARβ2, in nasopharyngeal carcinoma (NPC). To further explore targets contributing to aberrant retinoid signaling in NPC, the expression of cellular retinol-binding proteins (CRBPs), cellular retinoic acid-binding proteins (CRABPs), RARs, and retinoid X receptors (RXRs) was examined. Apart from RARβ2, transcriptional silencing of two CRBPs, CRBPI and CRBPIV, was observed in NPC cell lines and xenografts. Hypermethylation of CRBPI and CRBPIV CpG islands was found to be closely correlated with the loss of expression. Treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, resulted in reexpression of CRBPI and CRBPIV gene expression in NPC cell lines. Both CRBPI and CRBPIV hypermethylations were also observed in 43/48 (87.8%) and 26/48 (54.2%) primary NPC tumors, respectively. Here, we reported for the first time that CRBPIV was transcriptionally inactivated by promoter hypermethylation in human cancer. Simultaneous methylation of CRBPI, CRBPIV, and RARβ2 was commonly found in NPC primary tumors. Our findings implied that epigenetic disruption of the CRBPs, CRBPI and CRBPIV, is important in NPC tumorigenesis and may contribute to the loss of retinoic acid responsiveness in cancer. [ABSTRACT FROM AUTHOR]

• Haematococcus lacustris produces rich amounts of astaxanthin (AXT). • AXT production was improved due to cellular physiology changes induced by pH shock. • Gene expressions related to astaxanthin production were increased upon with pH shock. • Other inductive techniques could be combined to increase AXT production. In this study, the use of pH shock to improve astaxanthin synthesis in Haematococcus lacustris was investigated. It has been found that pH shock (pH = 4.5, 60 s) imposes stress in the cells and induces physiological changes, which result in astaxanthin accumulation. The optimal acid-base combination of pH shock was H 2 SO 4 -KOH, which increased the astaxanthin content per cell to 39 ± 6.92% than those of the control. In addition, pH shock can be applied simultaneously with the other inductive strategies such as high irradiance and carbon source supply. When high irradiance was applied simultaneously with pH shock, astaxanthin yield was increased 65 ± 0.541% than control. In addition, astaxanthin content per cell was increased 105 ± 6.66% than those of the control, with the concomitant application of carbon source addition with pH shock. Herein, these novel findings provide a useful technique for producing astaxanthin using H. lacustris. [ABSTRACT FROM AUTHOR]