The Physiological Function of nNOS-Associated CAPON Proteins and the Roles of CAPON in Diseases.

In this review, the structure, isoform, and physiological role of the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) are summarized. There are three isoforms of CAPON in humans, including long CAPON protein (CAPON-L), short CAPON protein (CAPON-S), and CAPON-S' protein. CAPON-L includes three functional regions: a C-terminal PDZ-binding motif, carboxypeptidase (CPE)-binding region, and N-terminal phosphotyrosine (PTB) structural domain. Both CAPON-S and CAPON-S' only contain the C-terminal PDZ-binding motif. The C-terminal PDZ-binding motif of CAPON can bind with neuronal nitric oxide synthase (nNOS) and participates in regulating NO production and neuronal development. An overview is given on the relationship between CAPON and heart diseases, diabetes, psychiatric disorders, and tumors. This review will clarify future research directions on the signal pathways related to CAPON, which will be helpful for studying the regulatory mechanism of CAPON. CAPON may be used as a drug target, which will provide new ideas and solutions for treating human diseases.

Dietary iron affects lipid deposition, nutritional element, and muscle quality in coho salmon (Oncorhynchus kisutch).

We investigated effect of dietary iron (Fe) on the lipid deposition, nutritional element, and muscle quality in coho salmon. Four level Fe diets at 23.7, 46.4, 77.3, and 127.7 mg/kg were fed to the post-larval coho salmon for 12 weeks. Our results showed that dietary Fe decreased the content of triglyceride and the activity of fatty acid synthetase, ATP-citrate lyase, and acetyl-CoA carboxylase. The content of Fe in muscle was increased with increasing dietary Fe levels, and dietary Fe affected the content of nutritional elements. In addition, dietary Fe levels affected the composition of fatty acids and the content of free amino acids, and increased muscle fiber size. The lower dietary Fe levels also affected the hardness, chewiness, resilience, springiness, cohesiveness, and gumminess of salmon muscle. In all, dietary Fe inhibited the lipid deposition and affected the content of nutritional element and muscle quality in coho salmon.

Roles of ubiquitination in the crosstalk between tumors and the tumor microenvironment (Review).

The interaction between a tumor and the tumor microenvironment (TME) plays a key role in tumorigenesis and tumor progression. Ubiquitination, a crucial post­translational modification for regulating protein degradation and turnover, plays a role in regulating the crosstalk between a tumor and the TME. Thus, identifying the roles of ubiquitination in the process may assist researchers to investigate the mechanisms underlying tumorigenesis and tumor progression. In the present review article, new insights into the substrates for ubiquitination that are involved in the regulation of hypoxic environments, angiogenesis, chronic inflammation­mediated tumor formation, and the function of cancer­associated fibroblasts and infiltrating immune cells (tumor­associated macrophages, T­cells, myeloid­derived suppressor cells, dendritic cells, and natural killer cells) are summarized. In addition, the potential targets of the ubiquitination proteasome system within the TME for cancer therapy and their therapeutic effects are reviewed and discussed.

Vitamin C regulates the production of reactive oxygen species through Wnt10b signaling in the gill of zebrafish.

In this study, the mechanism that vitamin C (VC) regulates the production of reactive oxygen species (ROS) through Wnt10b signaling was investigated in the gill of zebrafish (Danio rerio). The results showed that 0.5 and 1.0 g/kg VC diets induced the gene expression of Wnt10b, ß-catenin, SOD, CAT, and GSH-PX in gill. In addition, VC decreased the levels of H2O2, O2·- and ·OH, whereas the activities of SOD, CAT, and GSH-PX were increased by VC in the gill of zebrafish. To evaluate the role of Wnt10b in regulating oxidative stress, Wnt10b RNA was further interfered and the gene expression and activities of antioxidant enzymes were detected in gill. The result of Wnt10b RNA interference showed that Wnt10b signaling played a key role in regulating the gene expression of SOD, CAT, and GSH-PX. In all, VC may regulate the production of ROS through Wnt10b signaling in the gill of zebrafish (Danio rerio).

Evolution and Structure of API5 and Its Roles in Anti-Apoptosis.

Apoptosis, also named programmed cell death, is a highly conserved physiological mechanism. Apoptosis plays crucial roles in many life processes, such as tissue development, organ formation, homeostasis maintenance, resistance against external aggression, and immune responses. Apoptosis is regulated by many genes, among which Apoptosis Inhibitor-5 (API5) is an effective inhibitor, though the structure of API5 is completely different from the other known Inhibitors of Apoptosis Proteins (IAPs). Due to its high expression in many types of tumors, API5 has received extensive attention, and may be an effective target for cancer treatment. In order to comprehensively and systematically understand the biological roles of API5, we summarized the evolution and structure of API5 and its roles in anti-apoptosis in this review.

Dietary vitamin E regulates the activity of antioxidant enzymes through Wnt10b signaling in the muscle of zebrafish.

Vitamin E (VE) plays a crucial role in regulating the physiological functions of animals. In the present study, the mechanism by which VE regulates the activities of antioxidant enzymes through Wnt10b signaling was investigated in the muscle of zebrafish. It was found that the gene expression of Wnt10b, ß-catenin, and PPARγ was induced, while the GSK-3ß expression was inhibited by 52.34 and 101.27 mg kg-1 VE treatments. The generation of a hydroxy radical (˙OH) and superoxide anion (O2˙-) and the content of hydrogen peroxide (H2O2) were decreased by VE treatments. However, the activities of superoxide dismutase (SOD), peroxidase (POD), and glutathione peroxidase (GSH-PX) were increased by 52.34 and 101.27 mg kg-1 VE treatments. In addition, the content of saturated fatty acids (SFA) was decreased, but that of polyunsaturated fatty acids (PUFA) was increased by VE treatment. To confirm the role of Wnt10b in regulating antioxidant functions, Wnt10b RNA was interfered in zebrafish fed with different concentrations of VE diets. The results showed that the GSK-3ß gene expression was induced but the ß-catenin expression was inhibited by Wnt10b RNA interference in the muscle of zebrafish. The levels of O2˙-, H2O2, and ˙OH were enhanced, but the activities of SOD, GSH-PX, and POD were decreased by the interference of Wnt10b RNA. In all, our results indicated that VE could induce the Wnt10b/ß-catenin signaling pathway, which may further regulate the activities of antioxidant enzymes in the muscle of zebrafish.

Matrix Metalloproteinases in Invertebrates.

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.

Ubiquitination of Nonhistone Proteins in Cancer Development and Treatment.

Ubiquitination, a crucial post-translation modification, regulates the localization and stability of the substrate proteins including nonhistone proteins. The ubiquitin-proteasome system (UPS) on nonhistone proteins plays a critical role in many cellular processes such as DNA repair, transcription, signal transduction, and apoptosis. Its dysregulation induces various diseases including cancer, and the identification of this process may provide potential therapeutic targets for cancer treatment. In this review, we summarize the regulatory roles of key UPS members on major nonhistone substrates in cancer-related processes, such as cell cycle, cell proliferation, apoptosis, DNA damage repair, inflammation, and T cell dysfunction in cancer. In addition, we also highlight novel therapeutic interventions targeting the UPS members (E1s, E2s, E3s, proteasomes, and deubiquitinating enzymes). Furthermore, we discuss the application of proteolysis-targeting chimeras (PROTACs) technology as a novel anticancer therapeutic strategy in modulating protein target levels with the aid of UPS.

Identification and characterization of an atypical RIG-I encoded by planarian Dugesia japonica and its essential role in the immune response.

Retinoic acid-inducible gene I (RIG-I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG-I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG-I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that DjRIG-I is an ancient and unique protein of the RIG-I-like receptors (RLRs). In situ hybridization analysis revealed that the DjRIG-I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over-expression of DjRIG-I in planarians. The molecular simulation demonstrated that DjRIG-I formed a large hole-structure for the docking of dsRNAs, and the pull-down assay confirmed the interaction between DjRIG-I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG-I-mediated IFN and P38 signaling pathway, TBK1, IRF-3, Mx, and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the DjRIG-I expression by RNAi, inhibited the PAMPs-induced over-expression, suggesting that DjRIG-I is a key player for downstream signaling events. These results indicate that DjRIG-I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria-induced innate immunity of planarian.

Identification and characterization of a TNF receptor-associated factor in Dugesia japonica.

The tumor necrosis factor (TNF) superfamily consists of a wide variety of inflammatory cytokine, including cell-bound and secreted proteins. These TNFs function through binding and activation of the TNF receptors for modulating TNF-associated intracellular signals. A set of mammalian TNF receptor-associated factors (TRAFs) that have emerged as the major signal transducers for the TNF receptor superfamily, play an important role in both adaptive and innate immunity. However, the existence of TRAFs and their biological functions in planarian are still unknown. In this study, a new member of TRAFs, DjTRAF2, was identified in planarian Dugesia japonica. Phylogenetic analysis revealed that DjTRAF2 could be a new member of the invertebrate TRAF2 family. Sequence analysis showed that the open reading frame of DjTRAF2 had 1353 bp in length and encoded a putative protein of 450 amino acids with a predicted molecular mass of ~51.8 kDa and an isoelectric point of 7.052. Whole-mount in situ hybridization showed that DjTRAF2 was predominantly expressed in adult and regenerative pharynx, which is an important immune organ of planarian. Quantitative real-time PCR revealed that the transcriptional level of DjTRAF2 was significantly up-regulated after induced by pathogen-associated molecular patterns (polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and ß-glucan), suggesting that DjTRAF2 is involved in the immune response against pathogen invasion. Collectively, these results demonstrated that DjTRAF2 might play important roles in the innate immunity of planarian.

Synergistic Activity of an Antimetabolite Drug and Tyrosine Kinase Inhibitors against Breast Cancer Cells.

Antimetabolite drugs, including the adenosine deaminase inhibitor cladribine, have been shown to induce apoptosis in a variety of cancer cells, and have been widely used in clinical trials of various cancers in conjunction with tyrosine kinase inhibitors (TKIs). Combination treatment with cladribine and gefitinib or dasatinib is expected to have a synergistic inhibitory effect on breast cancer cell growth. Our results demonstrated that the combination treatment had synergistic activity against human breast cancer (MCF-7) cells, enhanced G2/M cell arrest and reactive oxygen species (ROS) generation, and increased the loss of mitochondrial membrane potential and cell apoptosis. In addition, the combination treatment decreased Bcl-2 expression. Our results demonstrated that cladribine in combination with gefitinib or dasatinib exerted synergistic anticancer effects on MCF-7 cells by inducing cell cycle arrest, ROS production and apoptosis through the mitochondria-mediated intrinsic pathway.

Tumor Necrosis Factor-α, a Regulator and Therapeutic Agent on Breast Cancer.

The cell-mediated immunity and cytotoxic agents play a significant role on tumor cell apoptosis. Tumor necrosis factor-α (TNF-α) is an intricate linker between inflammation and cancer through mediating the process of apoptosis and cell-mediated immunity. A variety of evidences have confirmed the critical role of TNF-α on tumor migration, proliferation, matrix degradation, tumor metastasis, invasion, and angiogenesis. Through binding to receptors, TNF-α participates in activating multiple cell signaling cascades that link inflammation, survival and evolution towards breast cancer. TNF-α is an important agent for tumor biotherapy, but its clinical application is limited for its severe fatal systemic toxicity. The poly-lactic acid microspheres (PLAM) with intratumoral cytokine release hold tremendous potential for the immunotherapy of breast cancer, and TNF-α antagonists may offer therapeutic potential in solid tumors. In addition, TNF-α is related with the blockage of estrogen and progesterone receptors. For breast cancer treatment, it is necessary to understand the molecular signaling pathways that mediate TNF-α and the aggressive behavior of negative breast cancer. The aim of present review is to summarize the effect of TNF-α on breast cancer cells.

Tumour necrosis factor-α inhibits hepatic lipid deposition through GSK-3ß/ß-catenin signaling in juvenile turbot (Scophthalmus maximus L.).

In this study, the mechanism that TNFα inhibits lipid deposition through GSK-3ß/ß-catenin signaling was investigated in the liver of juvenile turbot (Scophthalmus maximus L.) by injection of TNFα or TNFα inhibitor pomalidomide (POM). It was found that TNFα inhibited the expression of GSK-3ß and induced ß-catenin expression. TNFα inhibited the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), as well as the activity of lipoprotein lipase (LPL) and fatty acid synthetase (FAS). In addition, the level of triglyceride (TG), total cholesterol (TC), nonesterified fatty acid (NEFA), and glycerol was decreased by TNFα treatment in the liver. In the plasma, the level of TG, TC, low density lipoprotein cholesterol (LDL-C), NEFA, and glycerol was decreased, but high density lipoprotein cholesterol (HDL-C) was increased by TNFα treatment. However, compared to TNFα, POM had the opposite effect on the biochemical indexes and genes related to lipid deposition in the liver. The results indicated that TNFα may regulate hepatic lipid metabolism and fat distribution through GSK-3ß/ß-catenin signaling as well as transcription factors PPARγ and C/EBPα in juvenile turbot.

Synergistic inhibitory effect of berberine and d-limonene on human gastric carcinoma cell line MGC803.

This study aims at evaluating the anticancer effects of berberine hydrochloride (berberine) and d-limonene, alone and in combination, on human gastric carcinoma cell line MGC803 to determine whether berberine and d-limonene work synergistically and elucidate their mechanisms. MGC803 cells were treated with berberine and d-limonene, alone and in combination, for 24-48 h. The inhibitory effects of these drugs on growth were determined by MTT assay. The combination index and drug reduction index were calculated with the Chou-Talalay method based on the median-effect principle. Flow cytometry and laser scanning confocal microscopy were employed to evaluate the effects of both drugs on cell-cycle perturbation and apoptosis, generation of reactive oxygen species (ROS), mitochondrial membrane potential, and expression of Bcl-2 and caspase-3 in MGC803 cells. Berberine or d-limonene alone can inhibit the growth of MGC803 cells in a dose- and time-dependent manner. Berberine and d-limonene at a combination ratio of 1:4 exhibited a synergistic effect on anti-MGC803 cells. The two drugs distinctly induced intracellular ROS generation, reduced the mitochondrial transmembrane potential (ΔΨm), enhanced the expression of caspase-3, and decreased the expression of Bcl-2. The combination of berberine and d-limonene showed more remarkable effects compared with drugs used singly in MGC803 cells. The combination of berberine and d-limonene exerted synergistic anticancer effects on MGC803 cells by cell-cycle arrest, ROS production, and apoptosis induction through the mitochondria-mediated intrinsic pathway.

Effect of cadmium on the extracellular Na⁺, K⁺, and Ca²âº in the gill and small intestine of goldfish Carassius auratus.

In this study, the toxic effect of cadmium on extracellular Na(+), K(+), and Ca(2+) in the gill and small intestine of goldfish Carassius auratus was determined with the technique of ion chromatograph. Two-way ANOVA indicated that the two factors (Cd(2+) treatment and time) and the interaction factor had significant effect on the level of Na(+), K(+), and Ca(2+) in the small intestine and gill. 1.0 mg/L Cd(2+) significantly increased Ca(2+) level in the small intestine, but Ca(2+) level in the gill was significantly decreased by 1.0 and 5.0 mg/L Cd(2+) at 24, 48, and 72 h. Na(+) and K(+) level in the small intestine and gill was increased by 1.0 mg/L Cd(2+) at three time points, but increased by 5.0 mg/L Cd(2+) at a certain different time. In addition, Na(+) level was significantly decreased by 5.0 mg/L Cd(2+) at 24 or 48 h in the small intestine and gill. The results indicated that Cd(2+) played an important role in regulating the level of Na(+), K(+), and Ca(2+) in the small intestine and gill of goldfish C. auratus. A method was constructed to investigate the extracellular Na(+), K(+) and Ca(2+) in the tissues of gold fish with ion chromatography.

Analysis of reactive oxygen species, Ca²+ , and Hsp70 in the gill and mantle of clams Ruditapes philippinarum exposed in cadmium.

In this study, the probes 2',7'-dichlorofluorescein diacetate (H2 DCF-DA) and Fluo-3 AM were used to investigate the instantaneous change of reactive oxygen species (ROS) and Ca(2+) in the gill and mantle of clams Ruditapes philippinarum exposed in 0.05 mg L(-1) Cd(2+) with the laser-scanning confocal microscopy. The results indicated that Ca(2+) level was declined in the gill and slightly increased in the mantle. The level of ROS was declined in the gill, while the oscillation of ROS level was observed in the mantle. These data revealed that Ca(2+) could stimulate mitochondrial activity and enhance the respiratory chain in the gill and mantle. In addition, the expression of Hsp70 was increased in the gill and mantle of clams exposed in 0.05 mg L(-1) Cd(2+) . The change of Ca(2+) and ROS level affected the expression of Hsp70 in the gill and mantle. An appropriate method was established to analyze the effects of Cd(2+) on ROS, Ca(2+) , and Hsp70 in the gill and mantle of clams with confocal microscopy. Both confocal microscopy and chemical fluorescent are valuable tools for measurement of time-dependent intracellular ROS and Ca(2+) signals.

The effect of curcumin on breast cancer cells.

Curcumin, which is extracted from the plant Curcuma longa, has been used in the therapeutic arsenal for clinical oncology. Curcumin has chemopreventive and antitumoral activities against some aggressive and recurrent cancers. The expressions and activities of various proteins, such as inflammatory cytokines and enzymes, transcription factors, and gene-products linked with cell survivals and proliferation, can be modified by curcumin. Moreover, curcumin decreases the toxic effect of mitomycin C. Though curcumin has shown highly cytotoxic to some cancer cell lines, curcumin is insoluble and instable in water. The solubility of curcumin could be enhanced by utilizing the solubilizing properties of rubusoside. In addition, the selective delivery of synthetic analogs or nanotechnology-based formulations of curcumin to tumors may improve the chemopreventive and chemotherapeutic effects. The focus of this short review is to describe how curcumin participates in antitumor processes in breast cancer cells.

Effect of norcantharidin on the human breast cancer Bcap-37 cells.

Norcantharidin (NCTD), a chemically modified form of cantharidin, is a potential anticancer drug. In this study, the effects of NCTD on the cellular viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage in the human breast cancer cell line Bcap-37 were investigated with confocal and fluorescence microscopy. The cell cycle was further analyzed using the CellQuest software of a Becton-Dickinson FACS flow cytometer. The results indicated that the cellular viability was decreased with the growing concentrations of NCTD and time exposure. Moreover, the fluorescence intensity of ROS was increased, whereas the MMP was decreased in Bcap-37 cells with the growing concentrations of NCTD. NCTD induced a dose-dependent DNA damage and reduced the G1 peak in Bcap-37 cells. The G2/M peak of Bcap-37 was also decreased by the higher concentration of NCTD.

The effects of cerium on the growth and some antioxidant metabolisms in rice seedlings.

INTRODUCTION: The aims of the present study are to investigate the effects of Ce(3+) on the growth and some antioxidant metabolisms in rice seedlings (Oryza sativa L. cv Shengdao 16). MATERIALS AND METHODS: The rice was treated with 0, 0.05, 0.1, 0.5, 1.0, and 1.5 mM Ce(3+), respectively. The growth index of rice was measured. The chlorophyll content; catalase, superoxide dismutase, and peroxidase activities; and the level of hydrogen peroxide (H(2)O(2)), superoxide anion (O (2) (·-) ), and malondialdehyde were assayed. The accumulation of Ce(3+) and the uptake of mineral nutrition elements were analyzed with ICP-SF-MS. RESULTS AND DISCUSSION: Hormetic effects of Ce(3+) on the growth and some antioxidant metabolisms were found in the roots and shoots of rice. The roots can accumulate a much higher content of Ce(3+) than shoots and Ce(3+) mainly located in the cell wall of roots. Moreover, the uptake of K, Mg, Ca, Na, Fe, Mn, Zn, Cu, and Mo in the roots and shoots was affected with the exposure of different Ce(3+) treatments, which indicated that Ce(3+) affected the nutritional status of roots and shoots and further affected the growth of rice. CONCLUSION: The appropriate amount of Ce(3+) improved the defense system and growth of rice. The roots can accumulate a much higher content of Ce(3+) than shoots. Moreover, the uptake of K, Mg, Ca, Na, Fe, Mn, Zn, Cu, and Mo in the roots and shoots was affected with the exposure of different Ce(3+) treatments.

Analysis of reactive oxygen species in the guard cell of wheat stoma with confocal microscope.

Recently, the laser-scanning confocal microscope has become a routine technique and indispensable tool for cell biological studies. Previous studies indicated that reactive oxygen species (ROS) were generated in tobacco epidermal cells with confocal microscope. In the present studies, the probe 2',7'-dichlorof luorescein diacetate (H2DCF-DA) was used to research the change of ROS in the guard cell of wheat stoma, and catalase (CAT) was used to demonstrate that ROS had been labeled. The laser-scanning mode of confocal microscope was XYT, and the time interval between two sections was 1.6351 s. Sixty optical sections were acquired with the laser-scanning confocal microscope, and CAT (60,000 U mg⁻¹) was added after four optical sections were scanned. Furthermore, the region of interest (ROI) was circled and the fluorescence intensity of ROS was quantified with Leica Confocal Software. The quantitative data were exported and the trend chart was made with software Excell. The results indicated that ROS were produced intracellularly in stomatal guard cells, and the quantified fluorescence intensity of ROS was declined with CAT added. It is a good method to research the instantaneous change of ROS in plant cells with confocal microscope and fluorescence probe H2DCF-DA.