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马氏珠母贝硫酸软骨素合成酶基因1（ChSy-1）的克隆及功能研究
No full text硫酸软骨素（chondroitin sulfate，CS）是普遍存在于动物软骨组织中的一类重要的酸性高分子粘性多糖，其基于其精细的生物学结构发挥着许多生理功能，它的合成受到六种合成酶的调控，其中相关研究已经表明，在这六种酶中，ChSy-1在CS主链的合成以及糖链的延伸上起到非常重要的作用，当其缺失或突变时，小鼠、斑马鱼和人类都会出现相关的病症。目前对于ChSy-1的研究主要集中在人类群体以及模式动物中，在贝类中还未有相关报道。马氏珠母贝又称合浦珠母贝（Pinctada fucata，P.fucata），属于软体动物门，瓣腮纲，珍珠贝目，珍珠贝科，珠母贝属，是生产海水珍珠的主要母贝。本论文根据已有的马氏珠母贝转录组，进行ChSy-1的克隆和功能研究，为深入开展ChSy-1在马氏珠母贝中的功能奠定基础。
硫酸软骨素（chondroitin sulfate，CS）是普遍存在于动物软骨组织中的一类重要的酸性高分子粘性多糖，其基于其精细的生物学结构发挥着许多生理功能，它的合成受到六种合成酶的调控，其中相关研究已经表明，在这六种酶中，ChSy-1在CS主链的合成以及糖链的延伸上起到非常重要的作用，当其缺失或突变时，小鼠、斑马鱼和人类都会出现相关的病症。目前对于ChSy-1的研究主要集中在人类群体以及模式动物中，在贝类中还未有相关报道。马氏珠母贝又称合浦珠母贝（Pinctada fucata，P.fucata），属于软体动物门，瓣腮纲，珍珠贝目，珍珠贝科，珠母贝属，是生产海水珍珠的主要母贝。本论文根据已有的马氏珠母贝转录组，进行ChSy-1的克隆和功能研究，为深入开展ChSy-1在马氏珠母贝中的功能奠定基础。
2. 组织表达分析表明，ChSy-1基因在马氏珠母贝的外套膜、闭壳肌、肝胰腺、足、性腺、鳃、心脏中均有表达；在不同发育时期，ChSy-1基因则是在极体期、2细胞期和32细胞期表达量较高。
3. 进行了活体免疫刺激实验以及插核实验，探究ChSy-1在免疫中的功能。免疫刺激实验结果表明，在注射了脂多糖（LPS）和聚肌胞苷酸（Poly I:C）两种免疫刺激物之后，ChSy-1的表达量在肝胰腺中，LPS组在12h和72h后与对照组有显著差异（p < 0.05），Poly I:C组在12h和48h后与对照组有显著差异（p < 0.05）；在外套膜中，ChSy-1的表达量在LPS组48h和72h后与对照组有极显著差异（p < 0.01），Poly I:C组在24h、48h和72h后与对照组有显著差异（p < 0.05）。在进行插核后，在血淋巴中，ChSy-1在第3、5和第7天表达量与第一天相比有了显著差异（p < 0.05）；在肝胰腺中，ChSy-1在第3天和第7天表达量与第一天相比有了显著差异（p < 0.05），说明ChSy-1参与了马氏珠母贝的免疫应答反应。
4. 在矿化功能研究中，检测了ChSy-1在外套膜不同部位的mRNA表达量，结果发现ChSy-1基因在马氏珠母贝的外套膜三个部分都有较高的表达，且三者间并无显著性差异（p > 0.05），推测其在马氏珠母贝棱柱层和珍珠层的生物矿化过程中都有重要的作用。缺刻实验中，ChSy-1在马氏珠母贝缺刻后在外套膜中的表达量呈显著的上升趋势，说明其参与了壳损伤后的修复过程。BMP2基因RNA干扰后，ChSy-1基因的表达量与对照组比较，有了极显著上升（p < 0.01）。已有研究证明BMP2可以影响壳的生物矿化进程，本实验中对BMP2基因成功干扰后，ChSy-1在外套膜中的表达量显著上升，说明ChSy-1参与了影响生物矿化的BMP信号通路
Activation and characterization of a cryptic gene cluster reveals a cyclization cascade for polycyclic tetramate macrolactams
No full textPolycyclic tetramate macrolactams (PTMs) are a growing class of natural products and are derived from a hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway. PTM biosynthetic gene clusters are conserved and widely distributed in bacteria, however, most of them remain silent. Herein we report the activation of a PTM gene cluster in marine-derived Streptomyces pactum SCSIO 02999 by promoter engineering and heterologous expression, leading to the discovery of six new PTMs, pactamides A-F (11-16), with potent cytotoxic activity upon several human cancer cell lines. In vivo gene disruption experiments and in vitro biochemical assays reveal a reductive cyclization cascade for polycycle formation, with reactions sequentially generating the 5, 5/5 and 5/5/6 carbocyclic ring systems, catalysed by the phytoene dehydrogenase PtmB2, the oxidoreductase PtmB1, and the alcohol dehydrogenase PtmC, respectively. Furthermore, PtmC was demonstrated as a bifunctional cyclase for catalyzing the formation of the inner five-membered ring in ikarugamycin. This study suggests the possibility of finding more bioactive PTMs by genome mining and discloses a general mechanism for the formation of 5/5/6-type carbocyclic rings in PTMs
BATHYCONCHOECIA INCISA SP NOV (MYODOCOPA, HALOCYPRIDIDAE), A NEW SPECIES OF OSTRACOD FROM THE NERITIC ZONE OF THE SOUTH CHINA SEA
No full textSpecimens previously identified as Bathyconchoecia paulula sensu Yin & Chen, 1991 and Bathyconchoecia galerita sensu Yin & Chen, 1991, together with two similar specimens, are reclassified as a new species, Bathyconchoecia incisa sp. nov., which we describe and illustrate in detail here. The present species is similar to Bathyconchoecia sagittarius and Bathyconchoecia kornickeri in having a shallow notch on the posterodorsal corner of the carapace, but can be easily distinguished from these species by the structure of the posterior margin of the carapace and the striated sculpturing on the surface of the valves. The specimens on which this new classification was based were collected at shallow depths (<125 m) off the east coast of Hainan Island, in the northern South China Sea and in the Zengmu (James) Shoal and adjacent areas in the southern South China Sea. B. incisa is likely to be a shallow-water species or has a wide bathymetric range
Assignment of parentage by microsatellite analysis in the endangered Brachymystax lenok tsinlingensis (Salmonidae)
No full textWe evaluated the use of microsatellite markers to identify parentage in Brachy mystax lenok tsinlingensis Li, 1966, an endangered salmonid. Computer simulations showed that when no parent information was available, or when information was only available from 1 parent, then exclusion probabilities ranged from 23.6 to 45.1% and from 25.9 to 52.4%, respectively; combined exclusion probabilities for the 8 loci investigated were 97.7 and 98.4%, respectively. However, a breeding experiment with known parental and filial information resulted in 91.28% of progeny being exclusively assigned to their correct parent pair. Our data demonstrate that microsatellitebased parentage assignment is a reliable means with which to obtain information pertaining to genealogical relationships and could therefore benefit genetic conservation of this species
c-Jun N-terminal kinase (JNK) is involved in immune defense against bacterial infection in Crassostrea hongkongensis
No full textc-Jun N-terminal kinase (JNK) is a universal and essential subgroup of the mitogen-activated protein kinase (MAPK) superfamily, which is highly conserved from yeast to mammals and functions in a variety of physiological and pathological processes. In this study, we report the first oyster JNK gene homolog (ChJNK) and its biological functions in the Hong Kong oyster Crassostrea hongkongensis. The ChJNK protein consists of 383 amino acids and contains a conserved serine/threonine protein kinase (S_TKc) domain with a typical TPY motif. Phylogenetic analysis revealed that ChJNK shared a close evolutionary relationship with Crassostrea gigas JNK. Quantitative RT-PCR analyses revealed broad expression patterns of ChJNK mRNA in various adult tissues and different embryonic and larval stages of C hongkongensis. When exposed to Vibrio alginolyticus or Staphylococcus haemolyticus, ChJNK mRNA expression levels were significantly up-regulated in the hemocytes and gills in a time-dependent manner. Additionally, sub cellular localization studies that ChJNK is a cytoplasm-localized protein, and that its overexpression could significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells. In summary, this study provided the first experimental demonstration that oysters possess a functional JNK that participates in host defense against bacterial infection in C. hongkongensis. (C) 2016 Elsevier Ltd. All rights reserved
Effects of salinity and copper co-exposure on copper bioaccumulation in marine rabbitfish Siganus oramin
No full textMarine fish living in estuaries and coastal areas commonly encounter the stress of both salinity and metal pollution. In this study, euryhaline rabbitfish Siganus oramitt were exposed to 50 mu g L-1 waterborne Cu or 300 mu g g(-1) dry wt dietary Cu at salinity 33 parts per thousand, 25 parts per thousand, 20 parts per thousand, 10 parts per thousand, and 5 parts per thousand for 30 days. The Cu accumulation in the liver (>20-fold increase) and intestine (>5-fold increase) significantly increased after either waterborne or dietary Cu acclimation. Moreover, Cu accumulation was further enhanced in the liver, intestine, plasma, and whole body of Cu-exposed fish at lower salinities. Similarly, the waterborne Cu uptake rate constants (k(u)s) were stable in the control at different salinities but increased significantly (2-4 times higher) after waterborne Cu exposure. Conversely, the dietary Cu assimilation efficiencies (AEs) were significantly lower in the dietary Cu-exposed fish (3-5%) than in the control fish (8-16%) at different salinities, suggesting that dietary Cu acclimation partially alleviated the dietary Cu uptake from the high-Cu diet. The Cu efflux rate constants (Ices) were comparable among all treatments as 0.060 -0.071 d(-1). The changes of Cu accumulation by different salinities and Cu exposure were well estimated by the biokinetic modeling. In summary, the present study indicates that rabbitfish can regulate Cu uptake and accumulation when acclimated to different salinities, but the Cu-exposed rabbitfish failed to prevent the elevation of Cu accumulation at low salinities. It therefore suggested that the concurrence of low salinity and high Cu exposure enhances the risks of Cu bioaccumulation and toxicity in rabbitfish. (C) 2016 Elsevier Ltd. All rights reserved
Brevianamides and Mycophenolic Acid Derivatives from the Deep-Sea-Derived Fungus Penicillium brevicompactum DFFSCS025
No full textFour new compounds (1-4), including two brevianamides and two mycochromenic acid derivatives along with six known compounds were isolated from the deep-sea-derived fungus Penicillium brevicompactum DFFSCS025. Their structures were elucidated by spectroscopic analysis. Moreover, the absolute configurations of 1 and 2 were determined by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Compound 9 showed moderate cytotoxicity against human colon cancer HCT116 cell line with IC50 value of 15.6 mu M. In addition, 3 and 5 had significant antifouling activity against Bugula neritina larval settlement with EC50 values of 13.7 and 22.6 mu M, respectively. The NMR data of 6, 8, and 9 were assigned for the first time
Structurally diverse secondary metabolites from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001 and their biological evaluation
No full textFive new compounds, including a cytotoxic dimeric isocoumarin, bipenicilisorin (1), a merosesquiterpenoid, yaminterritrem C (2), a citrinin dimer, penicitrinone F (3), a alkaloid, terremide D (4), and a delta-Valerolacton, (E)-4-(propen-1-yl)-5,6-dihydro-2H-pyran-2-one (5), along with ten known compounds (6-15) were isolated from a deep-sea-derived fungus Penicillium chrysogenum SCSIO 41001. Their structures and absolute configurations were elucidated by NMR spectra, MS, CD, optical rotation, X-ray crystallography, and compared with literature data. Biological evaluation results revealed that 1 exhibited significant cytotoxic activities against K562, A549, and Huh-7 cell lines with IC50 values of 6.78, 6.94, and 2.59 mu M, respectively. Compound 3 exhibited moderate inhibitory activity against EV71 with IC50 value of 14.50 mu M. In addition, 13 and 14 showed specific COX-2 inhibitory activities with IC50 values of 1.09 and 1.97 mu M, respectively. (C) 2017 Published by Elsevier B.V
Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis
No full textFor identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC) combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture. In the approach, the complex mixture was considered as the combination of two individual samples for isobolographic analysis: the target compound and the mixture with complete removal of the target compound (subtracted residue). The two samples were prepared by HSCCC, and were used for the calculation of the expected effect of their combination. Using this approach, three compounds representing the major peaks in the HPLC of the brine shrimp toxic extract from P. pinnata (brine shrimp lethality test (BST) LC50 36.5 mu g/mL), pinnatin (1), 3,7-Dimethoxy-3',4'-methylenedioxy flavone (2), and karanjin (3), were prepared from the extract, and were tested for their synergistic potency by BST. The two-phase solvent system containing n-hexane-ethyl acetate-MeOH-water (14:7:10:10, v/v/v/v) was selected for the one-step HSCCC separation according to the partition coefficient values (K). The extract was separated into seven fractions (Fr1-7) by HSCCC with a total mass recovery of 96.3%. Fr2, 4, and 6 were the peak fractions corresponding to compounds 3, 2, and 1, respectively. The purities and recoveries of the target compounds after the chromatographic analysis were 95.9%-97.5% and 92.2%-96.1%, respectively. The subtracted residue of each target compound was performed by recombining all HSCCC fractions except the fraction containing the target compound. Isobolographic analysis disclosed a significant synergistic effect between karanjin and its subtracted residue (potency ratio of 0.47), which gave clear evidence that the toxicity of the extract results from synergistic interactions, and karanjin was one of the synergists participating in the interaction. The other two compounds were excluded from the synergism because these two compounds showed additive effects with their subtracted residues. Karanjin was the first synergistic compound identified from P. pinnata
Antiviral peptides from marine gorgonian-derived fungus Aspergillus sp SCSIO 41501
No full textA new cyclic pentapeptide and three new linear peptides, namely, aspergillipeptides D-G (1-4), were isolated from a culture broth of marine gorgonian-derived fungus Aspergillus sp. SCSIO 41501. Their structures were elucidated by spectroscopic analysis, and their absolute configurations were confirmed by Marfey's method. Compounds 1 and 2 showed evident antiviral activity against herpes simplex virus type 1 (HSV-1) with IC50 values of 9.5 and 19.8 mu M under their non-cytotoxic concentrations against a Vero cell line, respectively, and 1 also had antiviral activity against acyclovir-resistant clinical isolates of HSV-1. (C) 2017 Elsevier Ltd. All rights reserved