丁苯酞对大鼠脑缺血再灌注损伤后细胞色素-C的影响

目的观察丁苯酞对大鼠脑缺血再灌注损伤后细胞色素-C(Cyt-C)的变化。方法Wistar大鼠,随机分为假手术组、模型组、NBP治疗组、NBP预防组、NBP预防治疗组,每组再分为缺血再灌注6h,12h,24h3个亚组。采用改良线栓法制作大鼠局灶性脑缺血再灌注模型,HE染色观察形态学变化,免疫组化法检测脑组织细胞Cyt-C变化,TUNEL法原位检测凋亡细胞。结果假手术组脑组织中可见少许Cyt-C表达和凋亡细胞,模型组Cyt-C表达和凋亡细胞数较假手术组显著增加,于6h达高峰,之后逐渐下降,各时间点与假手术组比较均有统计学意义(P<0.01);丁苯酞治疗组Cyt-C表达和凋亡细胞数均减少,各时间点与模型组比较有统计学意义(P<0.01);丁苯酞预防组与模型组比较有统计学意义(P<0.05);丁苯酞预防加治疗组较丁苯酞治疗组Cyt-C比表达和凋亡细胞数减少(P<0.05)。结论大鼠脑缺血再灌注损伤后给予丁苯酞治疗能下调Cyt-C表达,同时减少神经细胞凋亡,提示丁苯酞可能通过抑制神经细胞Cyt-C的释放起到减少细胞凋亡,保护神经元的作用。

宰后鸭肉骨骼肌细胞中凋亡的发生及其影响因素研究

探讨宰后鸭肉骨骼肌细胞中是否发生细胞凋亡以及哪些因素对其产生影响,为进一步用细胞凋亡机理阐明水禽类肉的成熟机理提供新的试验依据。分析检测宰后鸭胸肉和腿肉中Bcl-2、Bax、Caspase-3蛋白表达量和线粒体细胞色素-C含量变化、TUNEL法检测阳性凋亡细胞核数量变化以及宰后ATP代谢和pH值变化情况。结果显示,宰后鸭肉骨骼肌中Bax/Bcl-2蛋白表达量比值均显著上调(P<0.05),线粒体细胞色素-C含量均显著降低(P<0.05),Caspase-3酶原裂解为20 kDa左右的活化片断,宰后0.5～8 h骨骼肌中阳性凋亡细胞核数量均显著增多(P<0.05);ATP含量和pH值在宰后0.5～8 h均呈现显著下降趋势(P<0.05)。Bax促凋亡蛋白表达量的占优,线粒体细胞色素-C的快速释放,凋亡关键蛋白酶Caspase-3的活化和阳性凋亡细胞核数量的急剧增多,表明宰后鸭肉骨骼肌细胞中明显存在一个细胞凋亡的过程。ATP的大量消耗和pH值下调等因素为细胞凋亡提供一个必要的细胞内环境,且鸭肉骨骼肌凋亡程度可能于宰后8 h左右达到一个峰值。

过氧化氢对游离线粒体Cyt-c释放的影响

目的在线粒体水平探讨过氧化氢(H2O2)预处理对游离线粒体细胞色素c(Cyt-c)释放的影响,揭示缺血预适应(IPC)对缺血再灌注(IR)损伤的作用机制。方法分离大鼠肝脏线粒体,制成游离线粒体。将游离线粒体分为5组,(1)为对照(C)组、(2)^(5)组分别加入12.5、25、50和100μmol/L的Ca2+,刺激游离线粒体10 min,检测游离线粒体Cyt-c,胱氨酸酶激活剂(Smac)的释放量。建立游离线粒体IPC再灌注模型,将游离线粒体分为7组,C组、IR组、加H2O2组共7组,对加H2O2组分别加入2μL H2O2,终浓度分别为1、2、5、20和100μmol/L;在H2O2处理后,再用100μmol/L Ca2+对IR组、加H2O2组模拟再灌注,检测Cyt-c的释放量,再对IR组和H2O2终浓度为1和2μmol/L组检测心磷脂活性的变化。结果与C组比较,Ca2+浓度25、50和100μmol/L组Cyt-c、Smac的释放量显著增高(P<0.05)。与IR组比较,H2O2浓度1、2μmol/L组Cyt-c的释放量明显减少(P<0.05)。Cyt-c释放减少时可改变心磷脂的活性。结论低浓度H2O2预处理通过使Cyt-c与心磷脂结合得更加紧密,而使线粒体在高Ca2+(100μmol/L)刺激后Cyt-c释放量减少,阻滞了凋亡途径的发生,是IPC对缺血再灌注损伤发挥效应的关键因素。

The relationship between nNOS and Cyt-c in mitochondria of glioma

Objective： The aim of this study was to investigate the association between neuronal nitric oxide synthase （nNOS） and the expression level of Cytochrome C （Cyt-c） in mitochondria. Methods： The pathological diagnosis of glioma and tumor classification was by HE staining, and we use immunohistochemistry method to analyse the level of nNOS in different pathological grade glioma and the expression level of Cyt-c in mitochondria meanwhile. Results： The levels of nNOS were highest in grade Ⅲ tumors, moderate in grade Ⅱ tumors, and lowest different in grade I tumors. There was significant difference of the nNOS levels among different pathological grade tumors （P 〈 0.05）. Furthermore, the similar phenomenon was observed in the expression level of Cyt-c in mitochondria （P 〈 0.05）. Conclusion： The expression level of nNOS and Cyt-c in mitochondria was significantly related to the pathological grade of glioma.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

Essential oil of Curcuma wenyujin induces apoptosis in human hepatoma cells

AIM: To investigate the effects of the essential oil of Curcuma wenyujin (CWO) on growth inhibition and on the induction of apoptosis in human HepG2 cancer cells. METHODS: The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. DNA fragmentation was visualized by agarose gel electrophoresis. Cell cycle and mitochondrial transmembrane potential (△Ψm) were determined by flow cytometry (FCM). Cytochrome C immunostaining was evaluated by fluorescence microscopy. Caspase-3 enzymatic activity was assayed by the cleavage of Ac-DEVD-R110. Cleaved PARP and active caspase-3 protein levels were measured by FCM using BD? CBA Human Apoptosis Kit. RESULTS: Treatment with CWO inhibited the growth of HepG2 cells in a dose-dependent manner, and the IC50 of CWO was approximately 70 μg/mL. CWO was found to inhibit the growth of HepG2 cells by inducing a cell cycle arrest at S/G2. DNA fragmentation was evidentlyobserved at 70 μg/mL after 72 h of treatment. During the process, cytosolic HepG2 cytochrome C staining showed a markedly stronger green fluorescence than in control cells in a dose-dependent fashion, and CWO also caused mitochondrial transmembrane depolarization. Furthermore, the results clearly demonstrated that both, activity of caspase-3 enzyme and protein levels of cleaved PARP, significantly increased in a dose- dependent manner after treatment with CWO. CONCLUSION: CWO exhibits an antiproliferative effect in HepG2 cells by inducing apoptosis. This growth inhibition is associated with cell cycle arrest, cytochrome C translocation, caspase 3 activation, Poly- ADP-ribose polymerase (PARP) degradation, and loss of mitochondrial membrane potential. This process involves a mitochondria-caspase dependent apoptosis pathway. As apoptosis is an important anti-cancer therapeutic target, these results suggest a potential of CWO as a chemotherapeutic agent.

Inhibition of mitochondria responsible for the anti-apoptotic effects of melatonin during ischemia-reperfusion

Objective： To investigate a possible mechanism responsible for anti-apoptotic effects of melatonin and provide theoretical evidences for clinical therapy. Methods： lschemia-reperfusion mediated neuronal cell injury model was constructed in cerebellar granule neurons （CGNs） by deprivation of glucose, serum and oxygen in media. After ischemia, melatonin was added to the test groups to reach differential concentration during reperfusion. DNA fragmentation, mitochondrial transmembrane potential, mitochondrial cytochrome c release and caspase-3 activity were observed after subjecting cerebellar granule neurons to oxygen-glucose deprivation （OGD）. Results： The results showed that OGD induced typical cell apoptosis change, DNA ladder and apoptosis-related alterations in mitochondrial functions including depression of mitochondrial transmembrane potential （its maximal protection ratio was 73.26%） and release of cytochrome c （its maximal inhibition ratio was 42.52%） and the subsequent activation of caspase-3 （its maximal protection ratio was 59.32%） in cytoplasm. Melatonin reduced DNA damage and inhibited release of mitochondrial cytochrome c and activation of caspase-3. Melatonin can strongly prevent the OGD-induced loss of the mitochondria membrane potential. Conclusion： Our findings suggested that the direct inhibition of mitochondrial pathway might essentially contribute to its anti-apoptotic effects in neuronal ischemia-reperfiusion.

Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1

Intracellular reactive oxygen species （ROS） are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interac- tion between easpase-9 and apoptotic protease-activating factor 1 （Apaf-1） via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/ Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H2O2-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can medi- ate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.

Effects of α-mangostin on apoptosis induction of human colon cancer

AIM:To investigate the effect of α-mangostin on the growth and apoptosis induction of human colon cancer cells.METHODS:The three colorectal adenocarcinoma cell lines tested (COLO 205,MIP-101 and SW 620) were treated with α-mangostin to determine the effect on cell proliferation by MTT assay,cell morphology,chromatin condensation,cell cycle analysis,DNA fragmentation,phosphatidylserine exposure and changing of mitochondrial membrane potential.The molecular mechanisms of α-mangostin mediated apoptosis were further investigated by Western blotting analysis including activation of caspase cascade,cytochrome c release,Bax,Bid,p53 and Bcl-2 modifying factor.RESULTS:The highest inhibitory effect of α-mangostin on cell proliferation of COLO 205,MIP-101 and SW 620 were 9.74 ± 0.85 μg/mL,11.35 ± 1.12 μg/mL and 19.6 ± 1.53 μg/mL,respectively.Further study showed that α-mangostin induced apoptotic cell death in COLO 205 cells as indicated by membrane blebbing,chromatin condensation,DNA fragmentation,cell cycle analysis,sub-G1 peak (P < 0.05) and phosphatidylserine exposure.The executioner caspase,caspase-3,the initiator caspase,caspase-8,and caspase-9 were expressed upon treatment with α-mangostin.Further studies of apoptotic proteins were determined by Western blotting analysis showing increased mitochondrial cytochrome c release,Bax,p53 and Bmf as well as reduced mitochondrial membrane potential (P < 0.05).In addition,up-regulation of tBid and Fas were evident upon treatment with α-mangostin (P < 0.01).CONCLUSION:α-Mangostin may be effective as an anti-cancer agent that induced apoptotic cell death in COLO 205 via a link between extrinsic and intrinsic pathways.

Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

In 2008, a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao. The causative species was determined to be Enteromorpha prolifera （Ulva prolifera O. F. Miiller）, a familiar green macroalga along the coastline of China. Rapid accumulation of a large biomass of floating U. prolifera prompted research on different aspects of this species. In this study, we constructed a nonnormalized cDNA library from the thalli of U. prolifera and acquired 10072 high-quality expressed sequence tags （ESTs）. These ESTs were assembled into 3 519 nonredundant gene groups, including 1 446 clusters and 2 073 singletons. After annotation with the nr database, a large number of genes were found to be related with chloroplast and ribosomal protein, GO functional classification showed 1 418 ESTs participated in photosynthesis and 1 359 ESTs were responsible for the generation of precursor metabolites and energy. In addition, rather comprehensive carbon fixation pathways were found in U. prolifera using KEGG. Some stress-related and signal transduction-related genes were also found in this study. All the evidences displayed that U. prolifera had substance and energy foundation for the intense photosynthesis and the rapid proliferation. Phylogenetic analysis of cytochrome c oxidase subunit I revealed that this green-tide causative species is most closely affiliated to Pseudendoclonium akinetum （Ulvophyceae）.

Altered expression of mitochondrial related genes in the native Tibetan placents by mitochondrial cDNA array analysis

Objective： To explore the mechanism of native Tibetan fetuses adaptation to hypoxia, we tried to find the different expression genes about mitochondrial function in the native Tibetan placents. Methods： In this study, the placents of native Tibetan and the high-altitude Hart （ha-Hart） were collected. After the total RNA extraction, the finally synthesized cDNAs were hybridized to mitochondrial array to find the altered expression genes between them. Then, the cytochrome c oxidase 17 （Coxl7）, dynactin 2 （DCTN2, also known as p50）, and vascular endothelial growth factor receptor （VEGFR, also known as KDR） were chosen from the altered expression genes to further verify the array results using the SYBR Green real-time PCR. Because the altered expression genes （such as Cybb and Cox 17） in the array results related to the activities of COXI and COXIV, the placental mitochondria activities of COXI and COXIV were measured to find their changes in the hypoxia. Results： By a standard of≥1.5 or ≤0.67, there were 24 different expressed genes between the native Tibetan and the ha-Han placents, including 3 up-regulated genes and 21 down-regulated genes. These genes were related to energy metabolism, signal transduction, cell proliferation, electron transport, cell adhesion, nucleotide-excision repair. The array results of Cox17, DCTN2 and KDR were further verified by the real-time RT-PCR. Through the mitochondria respiration measurements, the activity of COXI in the native Tibetan placents were higher than that of ha-Han, there was no difference in COXIV activity between them. Conclusion： The altered mitochondrial related genes in the native Tibetan placents may have a role in the high altitude adaptation for fetuses through changing the activity of mitochondrial COX.

Genetic diversity and population structure of Eleutheronema rhadinum in the East and South China Seas revealed in mitochondrial COI sequences

Eleutheronema rhadinum is a potential commercial fisheries species and is subject to intense exploitation in China. Knowledge on the population structure of E. rhadinum in Chinese coastal waters, which is important for sustainable exploitation and proper resource management, is lacking. In the present study, the genetic diversity and population structure of E. rhadinum were evaluated using a 564-base pair fragment of the mitochondrial cytochrome c oxidase subunit I （COl） gene. A total of 76 specimens were collected from three localities around the East （Qidong and Zhoushan） and South China Seas （Zhuhai）. Among these individuals, nine polymorphic sites were detected and 1 l distinct haplotypes were defined. High levels ofhaplotype diversity （h=0.759i0.035） and low levels of nucleotide diversity （re=0.001 98i0.003 26） were observed in these populations, Hierarchical analysis of molecular variance （AMOVA） indicated that 96.72% of the genetic variation occurred within the populations, whereas 3.28% occurred among populations. No significant genealogical branches or clusters were recognized on the neighbor-joining tree. Intra-group variation among populations was significant （~0~t=0.032 85, P〈0.01）. These results suggest that E. rhadinum populations in the East and South China Seas have developed divergent genetic structures. Tests of neutral evolution and mismatch distribution suggest that E. rhadinum may have experienced a population expansion. The present study provides basic information for the conservation and sustainable exploitation of this species.

Oridonin induces apoptosis in gastric cancer through Apaf-1,cytochrome c and caspase-3 signaling pathway

AIM:To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro.METHODS:The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide assay.After treatment with 10 μg/mL oridonin for 24 h and 48 h,the cells were stained with acridine orange/ethidium bromide.The morphologic changes were observed under an inverted fluorescence microscope.DNA fragmen-tation(a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay.After treated with oridonin(0,1.25,2.5,5 and 10 μg/mL),HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis,and oridonin-induced apoptosis in HGC-27 cells was detected.After treatment with oridonin for 24 h,the effects of oridonin on expression of Apaf-1,Bcl-2,Bax,caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction(RT-PCR) and Western blotting.RESULTS:Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose-and time-dependent manner.The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h(1.25,2.5,5 and 10 μg/mL) were 1.78% ± 0.36%,4.96% ± 1.59%,10.35% ± 2.76% and 41.6% ± 4.29%,respectively,which showed a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%,21.57% ± 3.75%,30.31% ± 4.91% and 61.19% ± 5.81%,with a significant difference(P < 0.05).The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%,31.86% ± 3.86%,48.30% ± 4.16% and 81.80% ± 6.72%,with a significant difference(P < 0.05).Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining.After treatment with oridonin,the cells became round,shrank,and developed small buds around the nuclear membrane while forming apoptotic bodies.Lactate dehydrogenase(LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h,LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4%(P < 0.001).However,the change in the release of LDH caused by necrosis was insignificant,suggesting thatthe major cause of oridonin-induced HGC-27 cell death was apoptosis.Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls(P < 0.05).And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%,12.8% ± 2.53%,28.5% ± 4.23% and 49.6% ± 3.76%,which were in a dose-dependent manner(P < 0.05).After treatment for 24 h,DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dosedependent manner.RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3(0.917 ± 0.103 vs 0.357 ± 0.019,P < 0.05),cytochrome c(1.429 ± 0.111 vs 1.002 ± 0.014,P < 0.05),Apaf-1(0.688 ± 0.101 vs 0.242 ± 0.037,P < 0.05) and Bax(0.856 ± 0.101 vs 0.278 ± 0.027,P < 0.05)(P < 0.05),whereas down-regulated in Bcl-2(0.085 ± 0.012 vs 0.175 ± 0.030,P < 0.05).Western blotting analysis also confirmed this result.CONCLUSION:Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1,caspase-3 and cytochrome c,which are highly dependent upon the mitochondrial pathway.

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

Interaction Between Cytochrome c and the Hapten 2,4-Dinitro- fluorobenzene by Electrospray Ionization Mass Spectrometry

Allergic contact dermatitis is a delayed hypersensitivity reaction, which results from skin exposure to low molecular weight chemicals such as haptens. To clarify the pathogenic mechanism, electrospray ionization mass spectrometry （ESI-MS） and hydrogen/deuterium （H/D） exchange, as well as UV spectroscopy, were applied to determine the interaction between the model protein cytochrome c （cyt c） and the hapten 2,4- dinitro-fiuorobenzene （DNFB）. The ESI-MS results demonstrate that the conformation of cyt c can change from native folded state into partially unfolded state with the increase of DNFB. The equilibrium state H/D exchange followed by ESI-MS further confirms the above results. UV spectroscopy indicates that the strong- field coordination between iron of heme （prosthetic group） and His18 or Met80 of cyt c is not obviously affected by the hapten.

DNA barcode assessment of Ceramiales(Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales （Rhodophyta） were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene （LSU）, universal plastid amplicon （UPA）, and partial mitochondrial cytochrome c oxidase subunit I gene （COl）. No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Syrnphyocladia latiuscula, and Neosiphoniajaponica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD （Barcode of Life Data） Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might he a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H.japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

Effect of matrine on HepG2 cells: role of glutathione and cytochrome c

Objective: To investigate the death mode of human hepatoma cells exposed to matrine and the role of glutathione (GSH) and cytochrome c. Methods: The MTT test and Cell Death Detection ELISA were used to identify cell death mode and viability of cells exposed to matrine. The volume of intracellular GSH was detected by GSH reductase. Finally Western blotting was chosen to analyze the expression of cytochrome c and Caspase-9 in HepG2 cells treated by matrine. Results: The apop-totic cell death induced by matrine in Hep G2 cells dramatically increased in the time-, dose-dependent manner. Matrine can exhaust intracellular GSH effectively to change the redox state in cells. Furthermore it affect the cytotoxicity of matrine. Re-sults of Western blotting showed that matrine induced the release of cytochrome c from mitochondria to cytoplasm, and then stimulate the cleavage of Caspase-9 in a time-dependent manner. Conclusion: Matrine induced apoptosis in Hep G2 cells through the mitochondrial pathway, and oxidative stress via depletion of GSH is directly involved in the apoptotic process.

The complete mitochondrial genome of Triuncina daii(Lepidoptera:Bombycidae) and its phylogenetic implications

The bombycid moth, Triuncina daii Xing Wang ＆ Zolotuhin, 2015, plays an important role for analyzing the phylogenetic relationships of the family Bombycidae （Lepidoptera： Bombycoidea）. Here we first describe the complete mitochondrial genome （mitogenome） of T. daii, which includes thirteen protein-coding genes （PCGs）, twenty-two transfer RNA genes, two ribosomal RNA genes and an A＋T-rich region, and we find the mitogenome is 15,482 bp in length （GenBank no. KY091643）. The genes order and orientation in the T. daii mitogenome are similar to other sequenced lepidopteran species. Except for cox1, all of the PCGs started with ATN. Twelve PCGs stopped at TAA except for cox1 which stops at a single T. Thirteen PCGs of available species are used to demonstrate the inner phylogenetic relationships of Bombycoidea. The bombycid species form a monophyletic clade with a bootstrap value of 100% and a posterior probability of 1.00.

Genetic Comparison among Rhynchophorus ferrugineus and Four Rhynchophorus Species

Genetic comparison between Rhynchophorus ferrugineus （Coleoptera： Curculionidae： Rhynchophorinae） and other four Rhynchophorus spp. such as R. bilineatus, R. phoenicis, R. cruentatus and R. palmarum was investigated using partial sequences of Cytochrome c oxidase subunit I （COl） mitochondrial gene. The number ofpolymorphic sites ranged from 79 to 102 while the genetic distances ranged from 0.058 to 0.095. The number of polymorphic sites, genetic distances and phylogenetic relationships among R. ferrugineus and the other four tested species revealed that R.ferrugineus was more closely related to R. bilineatus while it was distantly related to R. palmarum. It is possible to rank the five tested species as follows： R.ferrugineus, R. bilineatus, R. phoenicis, R. cruentatus and R. palmarum. The result of this study is the first step of the revision of the uncertain taxonomic status and phylogenetic relationships among the Rhynchophorus genus. Further analysis should be performed using other Rhynchophorus species as well as other molecular markers.

Genetic identification of two species of Pleuronichthys by DNA barcoding

DNA barcoding is a new method for biological taxonomy, offering the ability to identify species from fragments in any life-history stage. Pleuronichthys cornutus and P. japonicus are two morphologically similar species. Pleuronichthys japonicus has never been found previously in China. However, in this study, we identified both species using DNA barcoding （cytochrome c oxidase subunit I （COI））, the mtDNA control region and cytochrome b. The results reveal that： l） intraspecific variation in the DNA barcode is much less than interspecific variation; 2） the two morphologically similar species were placed into separate clades distinguishable by high bootstrap values; 3） COI barcodes are more powerful for identifying the two species than the other two mtDNA fragments.