Cellular Synthesis Research Articles

Bisphenol A attenuation in natural microcosm: Contribution of ecological components and identification of transformation pathways through stable isotope tracing

Residues of bisphenol A (BPA) are ubiquitously detected in the surface water due to its widespread usage. This study systematically investigated the dissipation and kinetics of BPA under simulated hydrolysis, direct and indirect photolysis, bacterial degradation, microbial degradation and natural attenuation in microcosm. Structural equation modeling (SEM) by using partial least square method in path coefficient analysis suggested that the microbial degradation was the major factor involved in the natural attenuation of BPA. The potential transformation products were identified by using liquid chromatography high-resolution mass spectrometry (LC-HRMS) and stable isotope tracing technique by simultaneous performing gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography mass spectrometry (GC–MS). A total of fourteen including three novel transformation products of BPA were identified to indicate five possible pathways. An increased yield of labeled (δ13C) CO2 and detection of 13C-labeled phospholipid fatty acids (PLFAs) indicated the mineralization of BPA and possible utilization of BPA or its transformation products by microbes for cellular membrane synthesis, respectively.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays.

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

AbstractGlucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation.

The viral replication proteins of plus-stranded RNA viruses orchestrate the biogenesis of the large viral replication compartments, including the numerous viral replicase complexes, which represent the sites of viral RNA replication. The formation and operation of these virus-driven structures require subversion of numerous cellular proteins, membrane deformation, membrane proliferation, changes in lipid composition of the hijacked cellular membranes and intensive viral RNA synthesis. These virus-driven processes require plentiful ATP and molecular building blocks produced at the sites of replication or delivered there. To obtain the necessary resources from the infected cells, tomato bushy stunt virus (TBSV) rewires cellular metabolic pathways by co-opting aerobic glycolytic enzymes to produce ATP molecules within the replication compartment and enhance virus production. However, aerobic glycolysis requires the replenishing of the NAD+ pool. In this paper, we demonstrate the efficient recruitment of pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) fermentation enzymes into the viral replication compartment. Depletion of Pdc1 in combination with deletion of the homologous PDC5 in yeast or knockdown of Pdc1 and Adh1 in plants reduced the efficiency of tombusvirus replication. Complementation approach revealed that the enzymatically functional Pdc1 is required to support tombusvirus replication. Measurements with an ATP biosensor revealed that both Pdc1 and Adh1 enzymes are required for efficient generation of ATP within the viral replication compartment. In vitro reconstitution experiments with the viral replicase show the pro-viral function of Pdc1 during the assembly of the viral replicase and the activation of the viral p92 RdRp, both of which require the co-opted ATP-driven Hsp70 protein chaperone. We propose that compartmentalization of the co-opted fermentation pathway in the tombusviral replication compartment benefits the virus by allowing for the rapid production of ATP locally, including replenishing of the regulatory NAD+ pool by the fermentation pathway. The compartmentalized production of NAD+ and ATP facilitates their efficient use by the co-opted ATP-dependent host factors to support robust tombusvirus replication. We propose that compartmentalization of the fermentation pathway gives an evolutionary advantage for tombusviruses to replicate rapidly to speed ahead of antiviral responses of the hosts and to outcompete other pathogenic viruses. We also show the dependence of turnip crinkle virus, bamboo mosaic virus, tobacco mosaic virus and the insect-infecting Flock House virus on the fermentation pathway, suggesting that a broad range of viruses might induce this pathway to support rapid replication.

Change in inorganic phosphate physical state can regulate transcription

ABSTRACTInorganic phosphate (Pi), a ubiquitous metabolite, is involved in all major biochemical pathways. We demonstrate that, in vitro, MgHPO4 (the intracellular Pi form) at physiological concentrations can exist in a metastable supersaturated dissolved state or as a precipitate. We have shown that in solution, MgHPO4 strongly stimulates exonuclease nascent transcript cleavage by RNA polymerase. We report here that MgHPO4 precipitate selectively and efficiently inhibits transcription initiation in vitro. In view of the MgHPO4 solubility and in vitro sensitivity of RNA synthesis to MgHPO4 precipitate, at physiological concentrations, MgHPO4 should cause a 50–98% inhibition of cellular RNA synthesis, thus exerting a strong regulatory action. The effects of Pi on transcription in vivo will, therefore, reflect the physical state of intracellular Pi.

Biological Stoichiometry Regulates Toxin Production in Microcystis aeruginosa (UTEX 2385).

Harmful algal blooms (HABs) are increasing in magnitude, frequency, and duration globally. Even though a limited number of phytoplankton species can be toxic, they are becoming one of the greatest water quality threats to public health and ecosystems due to their intrinsic toxicity to humans and the numerous interacting factors that undermine HAB forecasting. Here, we show that the carbon:nitrogen:phosphorus (C:N:P) stoichiometry of a common toxic phytoplankton species, Microcystis, regulates toxin quotas during blooms through a tradeoff between primary and secondary metabolism. Populations with optimal C:N (< 8) and C:P (< 200) cellular stoichiometry consistently produced more toxins than populations exhibiting stoichiometric plasticity. Phosphorus availability in water exerted a strong control on population biomass and C:P stoichiometry, but N availability exerted a stronger control on toxin quotas by regulating population biomass and C:N:P stoichiometry. Microcystin-LR, like many phytoplankton toxins, is an N-rich secondary metabolite with a C:N stoichiometry that is similar to the optimal growth stoichiometry of Microcystis. Thus, N availability relative to P and light provides a dual regulatory mechanism that controls both biomass production and cellular toxin synthesis. Overall, our results provide a quantitative framework for improving forecasting of toxin production during HABs and compelling support for water quality management that limit both N and P inputs from anthropogenic sources.

ALDH1A2 Is a Candidate Tumor Suppressor Gene in Ovarian Cancer.

Aldehyde dehydrogenase 1 family member A2 (ALDH1A2) is a rate-limiting enzyme involved in cellular retinoic acid synthesis. However, its functional role in ovarian cancer remains elusive. Here, we found that ALDH1A2 was the most prominently downregulated gene among ALDH family members in ovarian cancer cells, according to complementary DNA microarray data. Low ALDH1A2 expression was associated with unfavorable prognosis and shorter disease-free and overall survival for ovarian cancer patients. Notably, hypermethylation of ALDH1A2 was significantly higher in ovarian cancer cell lines when compared to that in immortalized human ovarian surface epithelial cell lines. ALDH1A2 expression was restored in various ovarian cancer cell lines after treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine. Furthermore, silencing DNA methyltransferase 1 (DNMT1) or 3B (DNMT3B) restored ALDH1A2 expression in ovarian cancer cell lines. Functional studies revealed that forced ALDH1A2 expression significantly impaired the proliferation of ovarian cancer cells and their invasive activity. To the best of our knowledge, this is the first study to show that ALDH1A2 expression is regulated by the epigenetic regulation of DNMTs, and subsequently that it might act as a tumor suppressor in ovarian cancer, further suggesting that enhancing ALDH1A2-linked signaling might provide new opportunities for therapeutic intervention in ovarian cancer.

Dynamically Tunable, Macroscopic Molecular Networks Enabled by Cellular Synthesis of 4-Arm Star-like Proteins

Summary Design of proteins with nonlinear topologies has emerged as a nascent branch of protein engineering, but significant applications remain to be seen. Here, we demonstrate the cellular synthesis of (SpyCatcher)4GFP, a 4-arm star-like protein enabled by spontaneous split GFP reconstitution, which further led to the creation of various protein networks exhibiting tunable mechanics and suitability for cell encapsulation. A derivative 4-arm star-like protein, (CarHC)4GFP, resulting from the conjugation of (SpyCatcher)4GFP with the SpyTag-fusion CarHC photoreceptors, can undergo rapid sol-gel and gel-sol transitions in response to AdoB12 and light, respectively. The chemo- and photo-induced phase transitions enabled encapsulation and controlled release of protein molecules such as the biofilm-degrading glycosyl hydrolase PslG, a potential agent for combatting multidrug-resistant bacterial species in chronic infections. The creation of those uncommon protein architectures promises great opportunities for materials biology and “smart” therapeutic delivery.

Creatine as a Candidate to Prevent Statin Myopathy.

Statins prevent cardiovascular diseases, yet their use is limited by the muscle disturbances they cause. Rarely, statin-induced myopathy is autoimmune, but more commonly it is due to direct muscle toxicity. Available evidence suggests that statin-induced creatine deficiency might be a major cause of this toxicity, and that creatine supplementation prevents it. Statins inhibit guanidinoacetate methyl transferase (GAMT), the last enzyme in the synthesis of creatine; thus, they decrease its intracellular content. Such decreased content could cause mitochondrial impairment, since creatine is the final acceptor of the phosphate group of adenosine triphosphate (ATP) at the end of mitochondrial oxidative phosphorylation. Decreased cellular synthesis of ATP would follow. Accordingly, ATP synthesis is decreased in statin-treated cells. In vitro, creatine supplementation prevents the opening of the mitochondrial permeability transition pore that is caused by statins. Clinically, creatine administration prevents statin myopathy in statin-intolerant patients. Additional research is warranted to hopefully confirm these findings. However, creatine is widely used by athletes with no adverse events, and has demonstrated to be safe even in double-blind, placebo-controlled trials of elderly individuals. Thus, it should be trialed, under medical supervision, in patients who cannot assume statin due to the occurrence of muscular symptoms.

Damage and Phenotype Change in PC12 Cells Induced by Lipopolysaccharide Can Be Inhibited by Antioxidants Through Reduced Cytoskeleton Protein Synthesis.

The present study investigated changes in cellular phenotype and oxidative stress during the inflammatory response in PC12 cells stimulated by lipopolysaccharide (LPS) and assessed the effects of minocycline, astragalus (AST), and baicalin on inflammation. PC12 cells were exposed to LPS with or without minocycline, AST, or baicalin. Cell viability was measured by a thiazolyl blue tetrazolium bromide (MTT) assay. Contrast and laser confocal microscopy were used to analyze changes in cellular phenotype and cytoskeleton synthesis. Western blotting tested the expression of α7nAChR and vimentin. Inhibitory ratio of superoxide dismutase (SOD) activity and leakage of lactate dehydrogenase (LDH) were detected to evaluate cellular oxidative stress. Results showed that LPS could attenuate PC12 cell viability in a time- and dose-dependent manner, which could be rescued by minocycline. In addition, minocycline could reverse PC12 cell phenotypic change and the synthesis of the mesenchymal cytoskeleton protein vimentin, both induced by LPS. During LPS-initiated inflammation, α7nAChR and vimentin expression were obviously inhibited by minocycline, AST, or baicalin. The inhibitory rate of SOD activity and LDH leakage in PC12 cells were increased by LPS and attenuated significantly when exposed to minocycline, AST, or baicalin. These findings suggest phenotype change, altered cytoskeleton protein synthesis, and oxidative stress are all involved in the inflammatory response in PC12 cells during which α7 nicotinic acetylcholine receptor (α7nAChR) is induced by LPS stimulation. Minocycline, AST, and baicalin have a protective effect against PC12 cell injury, acting as antioxidants and inhibitors of mesenchymal proteins.

Lessons learned from intervertebral disc pathophysiology to guide rational design of sequential delivery systems for therapeutic biological factors.

Intervertebral disc (IVD) degeneration has been associated with low back pain, which is a major musculoskeletal disorder and socio-economic problem that affects as many as 600 million patients worldwide. Here, we first review the current knowledge of IVD physiology and physiopathological processes in terms of homeostasis regulation and consecutive events that lead to tissue degeneration. Recent progress with IVD restoration by anti-catabolic or pro-anabolic approaches are then analyzed, as are the design of macro-, micro-, and nano-platforms to control the delivery of such therapeutic agents. Finally, we hypothesize that a sequential delivery strategy that i) firstly targets the inflammatory, pro-catabolic microenvironment with release of anti-inflammatory or anti-catabolic cytokines; ii) secondly increases cell density in the less hostile microenvironment by endogenous cell recruitment or exogenous cell injection, and finally iii) enhances cellular synthesis of extracellular matrix with release of pro-anabolic factors, would constitute an innovative yet challenging approach to IVD regeneration.

Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling.

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

Arg-8 of yeast subunit e contributes to the stability of F-ATP synthase dimers and to the generation of the full-conductance mitochondrial megachannel

The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.

Integrative roles of microRNAs in lipid metabolism and dyslipidemia.

The purpose of the review is to discuss recent advances in microRNA (miRNA) regulation of lipid metabolism and highlight the importance of miRNA-mediated gene regulation in dyslipidemia and fatty liver disease. This article reviews examples of miRNAs that bridge disparate metabolic pathways in the liver. For example, we highlight miRNAs that are regulated by the sterol-sensing pathway in the liver that in turn regulate cellular or systemic cholesterol, fatty acid, and glucose levels. The most widely studied of these miRNAs are miR-33a/b; however, we recently reported that miRNAs in the miR-183/96/182 cluster are also likely regulated by hepatic cholesterol content and mediate the observed glucose-lowering effects of the bile acid sequestrant colesevelam through the sterol-sensing pathway. In addition, several other hepatic and adipose miRNAs have been recently demonstrated to be key regulators of cellular lipid synthesis, storage, and catabolism, as well as systemic lipid metabolism. Moreover, many of these miRNAs are altered in fatty liver disease and dyslipidemia. miRNAs are not just fine-tuners of lipid metabolism, but critical regulatory factors in lipid homeostasis and health. Loss of these miRNA regulatory modules very likely contributes to the underlying metabolic defects observed in lipid disorders.

Sirt1 counteracts decrease in membrane phospholipid unsaturation and diastolic dysfunction during saturated fatty acid overload

BackgroundThe fatty acid (FA) composition of membrane phospholipid reflects at least in part dietary fat composition. Saturated FA (SFA) suppress Sirt1 activity, while monounsaturated FA (MUFA) counteract this effect. ObjectiveWe explored a role of Sirt1 in homeostatic control of the fatty acid composition of membrane phospholipid in the presence of SFA overload. Methods and resultsSirt1 deficiency in cardiomyocytes decreased the expression levels of liver X receptor (LXR)-target genes, particularly stearoyl-CoA desaturase-1 (Scd1), a rate-limiting enzyme in the cellular synthesis of MUFA from SFA, increased membrane SFA/MUFA ratio, and worsened left ventricular (LV) diastolic function in mice fed an SFA-rich high fat diet. In cultured cardiomyocytes, Sirt1 knockdown (KD) exacerbated the palmitate overload-induced increase in membrane SFA/MUFA ratio, which was associated with decrease in the expression of LXR-target genes, including Scd1. Forced overexpression of Scd1 in palmitate-overloaded Sirt1KD cardiomyocytes lowered the SFA/MUFA ratio. Nicotinamide mononucleotide (NMN) increased Sirt1 activity and Scd1 expression, thereby lowering membrane SFA/MUFA ratio in palmitate-overloaded cardiomyocytes. These effects of NMN were not observed for Scd1KD cardiomyocytes. LXRα/βKD exacerbated palmitate overload-induced increase in membrane SFA/MUFA ratio, while LXR agonist T0901317 alleviated it. NMN failed to rescue Scd1 protein expression and membrane SFA/MUFA ratio in palmitate-overloaded LXRα/βKD cardiomyocytes. The administration of NMN or T0901317 showed a dramatic reversal in membrane SFA/MUFA ratio and LV diastolic function in SFA-rich HFD-fed mice. ConclusionCardiac Sirt1 counteracted SFA overload-induced decrease in membrane phospholipid unsaturation and diastolic dysfunction via regulating LXR-mediated transcription of the Scd1 gene.

Hydrogen sulphide mitigates homocysteine-induced apoptosis and matrix remodelling in mesangial cells through Akt/FOXO1 signalling cascade

Cellular damage and accumulation of extracellular matrix (ECM) protein in the glomerulo-interstitial space are the signatures of chronic kidney disease (CKD). Hyperhomocysteinemia (HHcy), a high level of homocysteine (Hcy) is associated with CKD and further contributes to kidney damage. Despite a large number of studies, the signalling mechanism of Hcy-mediated cellular damage and ECM remodelling in kidney remains inconclusive. Hcy metabolizes to produce hydrogen sulphide (H2S), and a number of studies have shown that H2S mitigates the adverse effect of HHcy in a variety of diseases involving several signalling molecules, including forkhead box O (FOXO) protein. FOXO is a group of transcription factor that includes FOXO1, which plays important roles in cell growth and proliferation. On the other hand, a cell survival factor, Akt regulates FOXO under normal condition. However, the involvement of Akt/FOXO1 pathway in Hcy-induced mesangial cell damage remains elusive, and whether H2S plays any protective roles has yet to be clearly defined. We treated mouse mesangial cells with or without H2S donor, GYY4137 and FOXO1 inhibitor, AS1842856 in HHcy condition and determined the involvement of Akt/FOXO1 signalling cascades. Our results indicated that Hcy inactivated Akt and activated FOXO1 by dephosphorylating both the signalling molecules and induced FOXO1 nuclear translocation followed by activation of the FOXO1 transcription factor. These led to the induction of cellular apoptosis and synthesis of excessive ECM protein, in part, due to increased ROS production, loss of mitochondrial membrane potential (ΔΨm), reduction in intracellular ATP concentration, increased MMP-2, -9, -14 mRNA and protein expression, and Col I, IV and fibronectin protein expression. Interestingly, GYY4137 or AS1842856 treatment prevented these changes by modulating Akt/FOXO1 axis in HHcy. We conclude that GYY4137 and/or AS1842856 mitigates HHcy induced mesangial cell damage and ECM remodelling by regulating Akt/FOXO1 pathway.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production. Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.

The toxic mechanisms of BDE-47 to the marine diatom Thalassiosira pseudonana-a study based on multiple physiological processes

Polybrominated diphenyl ethers (PBDEs), a series of highly persistent organic pollutants (POPs), are ubiquitous in marine ecosystems. As key primary producers, microalgae are of great importance on evaluating the environmental outcome of PBDEs pollution. In this study, the toxic mechanisms of BDE-47 on the marine diatom Thalassiosira pseudonana were evaluated by measuring multiple physiological processes. Three concentrations of BDE-47 (25, 15 and 5 μg L−1) were used along with two controls (blank: no BDE-47 or DMSO; negative control: only DMSO). Experiments lasted 144 h (6 days), in which the actual BDE-47 concentrations, cell densities, nutrient (nitrate and phosphate) uptake, pigment compositions, photosynthetic physiology, cell morphology and cellular contents (organic carbon and nitrogen) were measured at 12–48 h intervals. The toxic mechanisms of BDE-47 on T. pseudonana cells were evaluated by measuring multiple physiological processes including photosynthesis, nutrient uptake, cellular material synthesis and cell cycle progressions. The cell divisions of T. pseudonana were severely inhibited by the stress of BDE-47, but the photosynthetic parameters were much less declined and recovered earlier than the cell divisions in the same BDE-47 treatments. The unsuppressed uptake rates of nutrients, increased cell volume and cellular contents indicated the cellular material synthesis proceeded normally. Finally, we found that the cell cycle was arrested in G2/M phase under the stress of BDE-47, we thus concluded that the inhibition of cell divisions by BDE-47 was not due to the lack of energy or cellular materials, where the cell cycle arrest happened; this might be the most important toxicological outcome.

Screening of the candidate genes related to low-temperature tolerance of Fenneropenaeus chinensis based on high-throughput transcriptome sequencing.

In order to screen the candidate genes of Fenneropenaeus chinensis related to low-temperature tolerance, this research takes juvenile prawns of F. chinensis (P40) in low temperature stress group (4°C) and normal temperature group (18°C) as experimental materials. The results showed that a total of 127,939 Unigenes with average length of 1,190 bp were obtained by assembly, of which 46% were annotated in the Nr database. A total of 1,698 differentially expressed genes were screened by differential gene expression analysis, of which 920 genes showed up-regulated expression and 778 genes showed down-regulated expression. Both GO and KEGG enrichment analysis revealed that differentially expressed genes were enriched in spliceosomes, ribosomes, bile secretion, ABC transport pathways, and cellular nitrogen compound synthesis. A further in-depth analysis obtained 8 genes that may be associated with low-temperature traits of F. chinensis. Five of them displayed up-regulated expression, including ATP-binding cassette protein C, acid ceramidase, glutathione transferase, C-type lectin and heat shock protein HSP70. The remaining three genes, γ-butyl betaine hydroxylase, β-hexosaminidase A and long chain fatty acid-CoA ligase displayed down-regulated expression. Eight differentially expressed genes were randomly selected and the real time RT-PCR verification showed that their expression levels were consistent with the sequencing results, demonstrating the accuracy of the sequencing results. The results of this study provide basic data for revealing the molecular mechanisms of F. chinensis in response to low temperature stress and the molecular assisted breeding of F. chinensis in low temperature.

Luteolin 7-Sulfate Attenuates Melanin Synthesis through Inhibition of CREB- and MITF-Mediated Tyrosinase Expression.

Antioxidants with antimelanogenic activity are potentially useful for the attenuation of skin hyperpigmentation disorders. In a previous study, luteolin 7-sulfate isolated from Phyllospadix iwatensis Makino, a marine plant, was shown to inhibit cellular melanin synthesis. The aim of the present study was to examine its action mechanism, focusing on the regulation of tyrosinase (TYR) expression in cells. Cell-based assay was undertaken using murine melanoma B16-F10 cells and primary human epidermal melanocytes (HEMs). Luteolin 7-sulfate showed lower toxicity compared to luteolin in B16-F10 cells. At the non-toxic concentration ranges, luteolin 7-sulfate attenuated melanin synthesis, stimulated by α-melanocyte-stimulating hormone or forskolin. Luteolin 7-sulfate attenuated forskolin-induced microphthalmia-associated transcription factor (MITF) and TYR expressions at the mRNA and protein levels in B16-F10 cells. It also attenuated the phosphorylation of cAMP-responsive element binding protein (CREB) stimulated by forskolin. Luteolin 7-sulfate also attenuated melanin synthesis in primary HEMs. This study demonstrates that luteolin 7-sulfate attenuates TYR gene expression through the intervention of a CREB- and MITF-mediated signaling pathway, leading to the decreased melanin synthesis.