Essential role of the Cytochrome P450 2 (CYP2) in the mud crab Scylla paramamosain antioxidant defense and immune responses.

Cytochrome P450 (CYPs) enzymes are one of the critical detoxification enzymes, playing a key role in antioxidant defense. However, the information of CYPs cDNA sequences and their functions are lacked in crustaceans. In this study, a novel full-length of CYP2 from the mud crab (designated as Sp-CYP2) was cloned and characterized. The coding sequence of Sp-CYP2 was 1479 bp in length and encoded a protein containing 492 amino acids. The amino acid sequence of Sp-CYP2 comprised a conserved heme binding site and chemical substrate binding site. Quantitative real-time PCR analysis revealed that Sp-CYP2 was ubiquitously expressed in various tissues, and it was highest in the heart followed by the hepatopancreas. Subcellular localization showed that Sp-CYP2 was prominently located in the cytoplasm and nucleus. The expression of Sp-CYP2 was induced by Vibrio parahaemolyticus infection and ammonia exposure. During ammonia exposure, ammonia exposure can induce oxidative stress and cause severely tissue damage. Knocking down Sp-CYP2 in vivo can increase malondialdehyde content and the mortality of mud crabs after ammonia exposure. All these results suggested that Sp-CYP2 played a crucial role in the defense against environmental stress and pathogen infection in crustaceans.

Quantitative Proteomic Analysis Reveals the Key Molecular Events Driving Phaeocystis globosa Bloom and Dissipation.

Phaeocystis globosa is a marine-bloom-forming haptophyte with a polymorphic life cycle alternating between free-living cells and a colonial morphotype, that produces high biomass and impacts ecological structure and function. The mechanisms of P. globosa bloom formation have been extensively studied, and various environmental factors are believed to trigger these events. However, little is known about the intrinsic biological processes that drive the bloom process, and the mechanisms underlying P. globosa bloom formation remain enigmatic. Here, we investigated a P. globosa bloom occurring along the Chinese coast and compared the proteomes of in situ P. globosa colonies from bloom and dissipation phases using a tandem mass tag (TMT)-based quantitative proteomic approach. Among the 5540 proteins identified, 191 and 109 proteins displayed higher abundances in the bloom and dissipation phases, respectively. The levels of proteins involved in photosynthesis, pigment metabolism, nitrogen metabolism, and matrix substrate biosynthesis were distinctly different between these two phases. Ambient nitrate is a key trigger of P. globosa bloom formation, while the enhanced light harvest and multiple inorganic carbon-concentrating mechanisms support the prosperousness of colonies in the bloom phase. Additionally, colonies in the bloom phase have greater carbon fixation potential, with more carbon and energy being fixed and flowing toward the colonial matrix biosynthesis. Our study revealed the key biological processes underlying P. globosa blooms and provides new insights into the mechanisms behind bloom formation.

Qian Yang Yu Yin Granule protects against hypertension-induced renal injury by epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Qian Yang Yu Yin Granule (QYYY) is a Chinese herbal formulation. It is used to treat hypertensive nephropathy for decades in China, but it is unknown that the exact mechanism of QYYY on hypertensive nephropathy. AIMS OF STUDY: The present study was to elucidate its epigenetic mechanism of QYYY on hypertensive nephropathy. MATERIALS AND METHODS: In the current study, HEK293T cells' proliferation induced by Ang II was chosen to observe epigenetic mechanisms of QYYY on renal damage. The cell proliferation was examined by MTT assays and ethynyldeoxyuridine analysis. Cell cycle analysis was performed. After treatment with QYYY, expression of Nicotinamide N-methyltransferase (NNMT), sirtuin1(SIRT1), S-adenosylhomocysteine(SAH), histone H3K4 methylation, and cortactin acetylation(acetyl-cortactin,ac-cortactin) were further investigated by western-blotting and real time PCR. DNA methylation was detected by ELISA. The study also observed the changes of SIRT1, SAH, H3K4 methylation, acetyl-cortactin when NNMT over-expressed by lentivirus transfection. Angiotensin II(Ang II) induced renal damage in spontaneously hypertensive rats(SHR). After eight weeks treatment of QYYY, blood pressure, serum and urine creatinine, and urinary microalbumin(mAlb) were assessed. The concentration of N1 -methylnicotinamide were detected by liquid chromatography with tandem mass spectrometry. The protein of NNMT, ac-cortactin, H3K3me3 were also assessed in vivo. RESULTS: QYYY inhibited HEK293T cells' proliferation, down-regulated the expression of NNMT, SAH, acetyl-cortactin and DNA methylation, up-regulated the expression of SIRT1, histone H3K4 trimethylation(H3K4me3). Over-expression of NNMT increased the expression of SAH and acetyl-cortactin, and reduced the expression of SIRT1 and H3K4me3. The study also demonstrated that QYYY promoted urinary creatinine excretion and reduced serum creatinine and urinary mAlb in SHR. QYYY decreased the concentration of N1 -methylnicotinamide in Ang II group. QYYY decreased the protein of NNMT, ac-cortactin and increased H3K4me3 in vivo. CONCLUSION: The results showed that QYYY alleviated renal impairment of SHR and inhibited HEK293T cells' proliferation induced by Ang II through the pathway of epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression, including histone methylation, DNA methylation and acetyl-cortactin. This study unveiled a novel molecular mechanism by which QYYY controlled the progression of hypertensive nephropathy.

iTRAQ-Based Quantitative Proteomic Analysis of a Toxigenic Dinoflagellate Alexandrium catenella at Different Stages of Toxin Biosynthesis during the Cell Cycle.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids that are produced mainly by marine dinoflagellates. PST biosynthesis in dinoflagellates is a discontinuous process that is coupled to the cell cycle. However, little is known about the molecular mechanism underlying this association. Here, we compared global protein expression profiles of a toxigenic dinoflagellate, Alexandrium catenella, collected at four different stages of toxin biosynthesis during the cell cycle, using an isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic approach. The results showed that toxin biosynthesis occurred mainly in the G1 phase, especially the late G1 phase. In total, 7232 proteins were confidently identified, and 210 proteins exhibited differential expression among the four stages. Proteins involved in protein translation and photosynthetic pigment biosynthesis were significantly upregulated during toxin biosynthesis, indicating close associations among the three processes. Nine toxin-related proteins were detected, and two core toxin biosynthesis proteins, namely, sxtA and sxtI, were identified for the first time in dinoflagellates. Among these proteins, sxtI and ompR were significantly downregulated when toxin biosynthesis stopped, indicating that they played important roles in the regulation of PST biosynthesis. Our study provides new insights into toxin biosynthesis in marine dinoflagellates: nitrogen balance among different biological processes regulates toxin biosynthesis, and that glutamate might play a key modulatory role.

iTRAQ-Based Quantitative Proteomic Analysis of a Toxigenic Dinoflagellate Alexandrium catenella and Its Non-toxigenic Mutant Exposed to a Cell Cycle Inhibitor Colchicine.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids mainly produced by marine dinoflagellates and their biosynthesis is associated with the cell cycle. Study shows that colchicine can cease cell division and inhibit PST production of dinoflagellates. However, the molecular mechanism behind this linkage is unknown. Here, we applied the iTRAQ-based proteomic approach to investigate protein expression profiles of a toxigenic dinoflagellate Alexandrium catenella (ACHK-T) and its non-toxigenic mutant (ACHK-NT) when treated with colchicine. The results showed that the cell cycles of both strains were arrested at the G1 phase by colchicine, and the toxin biosynthesis of ACHK-T was inhibited. Among 6,988 proteins identified, 113 and 253 proteins were differentially expressed in the colchicine-treated ACHK-T and ACHK-NT, respectively, compared with their non-colchicine treatments. Proteins involved in reactive oxygen species scavenging and protein degradation were upregulated in both strains while proteins participating in photosynthetic pigment biosynthesis and nitrogen metabolism presented different expressions. Nitrate reductase and glutamine synthetase were altered insignificantly in the colchicine-treated ACHK-T while both of them were remarkably downregulated in the colchicine-treated ACHK-NT, suggesting a feedback regulation between PST production and nitrogen metabolism in ACHK-T. Nitrogen originally for PST biosynthesis might be reallocated to photosynthetic pigment biosynthesis in the colchicine-treated ACHK-T. A total of 55 homologs of 7 toxin-related proteins were obtained; however, they altered insignificantly in both colchicine-treated strains, suggesting that toxin biosynthesis might be post-translationally regulated. Our study provided new insights into toxin biosynthesis in marine dinoflagellates.

Whole Transcriptomic Analysis Provides Insights into Molecular Mechanisms for Toxin Biosynthesis in a Toxic Dinoflagellate Alexandrium catenella (ACHK-T).

Paralytic shellfish toxins (PSTs), a group of neurotoxic alkaloids, are the most potent biotoxins for aquatic ecosystems and human health. Marine dinoflagellates and freshwater cyanobacteria are two producers of PSTs. The biosynthesis mechanism of PSTs has been well elucidated in cyanobacteria; however, it remains ambiguous in dinoflagellates. Here, we compared the transcriptome profiles of a toxin-producing dinoflagellate Alexandrium catenella (ACHK-T) at different toxin biosynthesis stages within the cell cycle using RNA-seq. The intracellular toxin content increased gradually in the middle G1 phase and rapidly in the late G1 phase, and then remained relatively stable in other phases. Samples from four toxin biosynthesis stages were selected for sequencing, and finally yielded 110,370 unigenes, of which 66,141 were successfully annotated in the known databases. An analysis of differentially expressed genes revealed that 2866 genes altered significantly and 297 were co-expressed throughout the four stages. These genes participated mainly in protein metabolism, carbohydrate metabolism, and the oxidation-reduction process. A total of 138 homologues of toxin genes were identified, but they altered insignificantly among different stages, indicating that toxin biosynthesis might be regulated translationally or post-translationally. Our results will serve as an important transcriptomic resource to characterize key molecular processes underlying dinoflagellate toxin biosynthesis.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. SCIENTIFIC QUESTION: Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. TECHNICAL SIGNIFICANCE: High-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. SCIENTIFIC SIGNIFICANCE: This review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms.

iTRAQ-based quantitative proteomic analysis of a toxigenic dinoflagellate Alexandrium catenella and its non-toxic mutant.

Paralytic shellfish toxins (PSTs) are a group of potent neurotoxic alkaloids produced by cyanobacteria and dinoflagellates. The PST biosynthesis gene cluster and several toxin-related proteins have been unveiled in cyanobacteria, yet little is known about dinoflagellates. Here, we compared the protein profiles of a toxin-producing dinoflagellate Alexandrium catenella (ACHK-T) and its non-toxic mutant (ACHK-NT), and characterized differentially displayed proteins using a combination of the iTRAQ-based proteomic approach and the transcriptomic database. Totally 3488 proteins were identified from A. catenella, and proteins involved in carbohydrate, amino acid and energy metabolism were the most abundant. Among them, 185 proteins were differentially displayed: proteins involved in amino acid biosynthesis, protein and carbohydrate metabolism and bioluminescence were more abundant in ACHK-T, while proteins participating in photosynthesis, fatty acid biosynthesis, and the processes occurring in peroxisome displayed higher abundances in ACHK-NT. Seven toxin-related proteins were identified but they varied insignificantly between the two strains. Different carbon and energy utilization strategies were potentially related to the toxin producing ability, and the regulation mechanism of PST biosynthesis was more complex in dinoflagellates. Our study provides the first comprehensive dataset on the dinoflagellate proteome and lays the groundwork for future proteomic study.

Comparative proteomics reveals highly and differentially expressed proteins in field-collected and laboratory-cultured blooming cells of the diatom Skeletonema costatum.

Diatoms are a major phytoplankton group causing extensive blooms in the ocean. However, little is known about the intracellular biological processes occurring during the blooming period. This study compared the protein profiles of field-collected and laboratory-cultured blooming cells of Skeletonema costatum, and identified highly and differentially expressed proteins using the shotgun proteomic approach. A total of 1372 proteins were confidently identified with two or more peptides. Among them, 222 and 311 proteins were unique to the laboratory and field samples respectively. Proteins involved in photosynthesis, translation, nucleosome assembly, carbohydrate and energy metabolism dominated the protein profiles in both samples. However, different features of specific proteins were also found: proteins participated in light harvesting, photosynthetic pigment biosynthesis, photoprotection, cell division and redox homeostasis were highly detected in the field sample, whereas proteins involved in translation, amino acid and protein metabolic processes, and nitrogen and carbon assimilation presented high detection rates in the laboratory sample. ATP synthase cf1 subunit beta and light harvest complex protein were the most abundant protein in the laboratory and field samples respectively. These results indicated that S. costatum had evolved adaptive mechanisms to the changing environment, and integrating field and laboratory proteomic data should provide comprehensive understanding of bloom mechanisms.

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen.

Nitrogen (N) is the major nutrient limiting phytoplankton growth and productivity over large ocean areas. Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the ocean. However, very little is known about their adaptive response to changing ambient N. Here, we compared the protein profiles of a marine dinoflagellate Prorocentrum donghaiense grown in inorganic N-replete, N-deplete and N-resupplied conditions using 2-D fluorescence differential gel electrophoresis. The results showed that cell density, chlorophyll a and particulate organic N contents presented low levels in N-deplete cells, while particulate organic carbon content and glutamine synthetase (GS) activity maintained high levels. Comparison of the protein profiles of N-replete, N-deplete and N-resupplied cells indicated that proteins involved in photosynthesis, carbon fixation, protein and lipid synthesis were down-regulated, while proteins participating in N reallocation and transport activity were up-regulated in N-deplete cells. High expressions of GS and 60 kDa chaperonin as well as high GS activity in N-deplete cells indicated their central role in N stress adaptation. Overall, in contrast with other photosynthetic eukaryotic algae, P. donghaiense possessed a specific ability to regulate intracellular carbon and N metabolism in response to extreme ambient N deficiency.

Comparative transcriptome analysis of a toxin-producing dinoflagellate Alexandrium catenella and its non-toxic mutant.

The dinoflagellates and cyanobacteria are two major kingdoms of life producing paralytic shellfish toxins (PSTs), a large group of neurotoxic alkaloids causing paralytic shellfish poisonings around the world. In contrast to the well elucidated PST biosynthetic genes in cyanobacteria, little is known about the dinoflagellates. This study compared transcriptome profiles of a toxin-producing dinoflagellate, Alexandrium catenella (ACHK-T), and its non-toxic mutant form (ACHK-NT) using RNA-seq. All clean reads were assembled de novo into a total of 113,674 unigenes, and 66,812 unigenes were annotated in the known databases. Out of them, 35 genes were found to express differentially between the two strains. The up-regulated genes in ACHK-NT were involved in photosynthesis, carbon fixation and amino acid metabolism processes, indicating that more carbon and energy were utilized for cell growth. Among the down-regulated genes, expression of a unigene assigned to the long isoform of sxtA, the initiator of toxin biosynthesis in cyanobacteria, was significantly depressed, suggesting that this long transcript of sxtA might be directly involved in toxin biosynthesis and its depression resulted in the loss of the ability to synthesize PSTs in ACHK-NT. In addition, 101 putative homologs of 12 cyanobacterial sxt genes were identified, and the sxtO and sxtZ genes were identified in dinoflagellates for the first time. The findings of this study should shed light on the biosynthesis of PSTs in the dinoflagellates.

Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae).

Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M) showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

[The effect of RNA interfering TLR4 signal pathway on phagocytosis of Kupffer cells].

OBJECTIVE: To investigate the effect of RNA interfering TLR4 signal pathway on phagocytosis of Kupffer cells. METHODS: RAW2647 mice mononuclear macrophage leukemia cells were observed. The tested group was interfered by Tlr4-mus-1567 RNA which had the best result confirmed by QPCR, cells interfered by Negative Control RNA as NC group, and normal cell as control. We perform the phagocytosis test on each group. RESULTS: The tested group has lower phagocytes percentage than control (17.67% +/- 3.51% vs 32.00% +/- 3.00%, P < 0.01), and lower phagocytic index (46.33% +/- 7.51% vs 82.00% +/- 6.08%, P < 0.01). CONCLUSIONS: Decreased phagocytic activity was observed on Kupffer cells by RNA interference.