Natural variation in the cytochrome c oxidase subunit 5B OsCOX5B regulates seed vigor by altering energy production in rice

Seed vigor is a crucial trait for the direct seeding of rice.Here we examined the genetic regulation of seed vigor traits in rice,including germination index(GI)and germination potential(GP),using a genome-wide association study approach.One major quantitative trait locus,qGI6/qGP6,was identified simultaneously for both GI and GP.The candidate gene encoding the cytochrome c oxidase subunit 5B(OsCOX5B)was validated for qGI6/qGP6.The disruption of OsCOX5B caused the vigor traits to be significantly lower in Oscox5b mutants than in the japonica Nipponbare wild type(WT).Gene co-expression analysis revealed that OsCOX5B influences seed vigor mainly by modulating the tricarboxylic acid cycle process.The glucose levels were significantly higher while the pyruvic acid and adenosine triphosphate levels were significantly lower in Oscox5b mutants than in WT during seed germination.The elite haplotype of OsCOX5B facilitates seed vigor by increasing its expression during seed germination.Thus,we propose that OsCOX5B is a potential target for the breeding of rice varieties with enhanced seed vigor for direct seeding.

Cancer and age related colonic crypt deficiencies in cytochrome c oxidase Ⅰ

AIM: To investigate whether defi ciency of expressionof cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer.METHODS: The pattern and level of expression of CcOI in non-neoplastic colonic crypts,and in dysplastic tissues,was assessed using standard immunohis-tochemical methods.Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason.Tissue samples were also obtained from surgical colonic resections.Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves.Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI.In most crypts the loss was apparent throughout the entire crypt,while in a small minority the loss was segmental.The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous.The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated.Data analyses were performed using SPSS statistical package 17.0.RESULTS: The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age,with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men.CcOI-DC increases after age 40 years,so that between the ages of 40 and 44 years the average frequency of CcOI- DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men.By 80-84 years of age,the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%.The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01.For women over age 60 years,deficiency of CcOI expression is greater in those women who have had a cancer in their colon.The frequency of CcOI-DC,measured in men,increased in tissues adjacent to colon cancer,being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 yearsand 14.13% ± 0.35% in resected histologically normal tissue of men with cancer in the same age range,P < 0.001.Similar signifi cant differences were noted in older age ranges.The frequency of CcOI-DC crypts in the cecum and sigmoid colon of an individual are signifi cantly correlated,with an R2 = 0.414 for women and R2 = 0.528 for men,P < 0.001.This suggests that the factors determining the level of CcOI deficiency act throughout the colon.Most defective crypts are in clusters of two or more,a likely consequence of crypt fission.In the non-neoplastic margins of cancers,crypts are frequently defi cient for CcOI,and such crypts may appear in large clusters,some containing more than 100 defi cient crypts.CcOI defi ciency is also apparent in colon cancers and sometimes involves a large section of the tumor.Overall,CcOI deficient cells can be visualized in segments of crypts,in whole crypts that increase in frequency with age,in crypts undergoing f ission,in clusters of crypts where the clusters increase in size with age,in increased frequency near tumors,in large clusters in the intimate margins of tumors,and in the tumors themselves.There is no clear dividing line between early stages that can be considered aspects of aging and later stages that can be considered aspects of the progression to cancer.This ambiguity may re ect a rather general situation leading to adult cancer where the early stages of cellular change appear to be relatively innocuous features of the aging process but over decades may evolve into malignancy.CONCLUSION: CcOI defi cient crypts increase in frequency with age,and clusters of defi cient crypts are associated with,and may give rise to,colon cancer.

Artesunate Effect on Schistosome Thioredoxin Glutathione Reductase and Cytochrome c Peroxidase as New Molecular Targets in Schistosoma mansoni-infected Mice

Objective To investigate the possible effect of artesunate （ART） on schistosome thioredoxin glutathione reductase （TGR） and cytochrome c peroxidase （CcP） in Schistosoma mansoni-infected mice. Methods A total of 200 laboratory bred male Swiss albino mice were divided into 4 groups （50 mice in each group）. Group I： infected untreated group （Control group） received a vehicle of 1% sodium carbonyl methylcellulose （CMC-Na）; Group II： infected then treated with artesunate; Group III infected then treated with praziquantel, and group IV： infected then treated with artesunate then praziquantel. Adult S. mansoni worms were collected by Animal Perfusion Method, tissue egg counted, TGR, and CcP mRNA Expression were estimated of in $. mansoni adult worms by semi-quantitative rt-PCR. Results Semi-quantitative rt-PCR values revealed that treatment with artesunate caused significant decrease in expression of schistosome TGR and CcP in comparison to the untreated group. In contrast, the treatment with praziquantel did not cause significant change in expression of these genes. The results showed more reduction in total worm and female worm count in combined ART-PZQ treated group than in monotherapy treated groups by either ART or PZO, Moreover, complete disappearance （100%） of tissue eggs was recorded in ART-PZQ treated group with a respective reduction rate of 95.9% and 68.4% in ART- and PZQ-treated groups. Conclusion The current study elucidated for the first time that anti-schistosomal mechanisms of artesunate is mediated via reduction in expression of schistosome TGR and CcP. Linking these findings, addition of artesunate to praziquantel could achieve complete cure outcome in treatment of schistosomiasis.

Effects of High Concentration Glucose on the Expression of NF-κB,Bax and Cytochrome C and Apoptosis of Islet Cells in Mice

The roles of NF-kappaB （NF-κB） expression, Bax activity and cytochrome C （Cyt C） release, apoptosis of islet cells induced by high concentration glucose were explored in vitro. Pancreatic islet cells, which were isolated from Kunming mice, were cultured with different concentrations of glucose in DMEM, and divided into the following groups： G1, G2, G3, G4, G5, and G6 groups, corresponding to the glucose concentrations of 5.6, 7.8, 11.1, 16.7, 22.5, and 27.6 mmol/L, respectively. After culture for 120 h, insulin secretion was evaluated by radioimmunoassay, and the NF-rd3 expression was detected by immunocytochemistry. Bax activity and Cyt C release were measured by immunofluorescence, and apoptosis was examined by Hoechst33342 assay. The results showed that in GI, G2 and G3 groups, insulin secretion was enhanced with the increase of glucose concentration, and the NF-κB expression was also increased （P〈0.05）, but Bax activity, Cyt C release and apoptosis rate showed no significant difference among them. However, in G4, G5, and G6 groups, apoptosis rate of islet cells, NF-rd3 expression, Bax activity, and Cyt C release were all significantly increased, and insulin secretion was impaired as compared with G1, G2, and G3 groups （P〈0.05）. It was concluded that the exposure of islet cells to high glucose could induce islet cells apoptosis as well as impaired insulin secretion. The NF-κB signaling pathway and mitochondria pathway in islet cells might play some roles in the progressive loss of islet cells in diabetes. The inhibition of the NF-κB expression could be an effective strategy for protecting pancreatic islet cells.

Interaction of methylenetetrahydrofolate reductase C677T,cytochrome P4502E1 polymorphism and environment factors in esophageal cancer in Kazakh population

AIM： To evaluate the association and interaction of genetic polymorphisms in methylenetetrahydrofolate reductase （MTHER） and cytochrome P4502E1 （CY- P4502E1）, environment risk factors with esophageal cancer （EC） in Kazakh, a high EC incidence area of Xinjiang Uygur Autonomous Region, China. METHODS： A 1：2 matched case-control study was conducted with 120 cases of EC and 240 populationor hospital-based controls. The controls were matched for sex, nationality, area of residence and age within a 5-year difference. MTHER and CYP4502E1 genotypes were identified by PCR-based restriction fragment length polymorphism （RFLP）. A conditional logistic regression model was established to identify risk factors. The strata method was adopted in interaction analysis. RESULTS： Low consumption of green vegetables and fresh fruits, alcohol drinking, and unsafe water （shallow well, or river） were found to be the risk factors for EC. Individuals with the MTHFR677 （C/T ＋ T/T） genotype had a 2.62-fold （95% CI： 1.61-4.28） risk of developing EC compared with those who carried the C/C genotype. Individuals with the CYP4502EIC1/C1 genotype had a 3.00-fold （95% CI： 1.82-4.96） risk compared with those who carried the CYP4502E1 （C1/C2 ＋ C2/C2） genotype. Gene-environment interaction analysis showed that MTHFR677 gene polymorphism was correlated with consumption of green vegetables and fresh fruit, while CYP4502E1 C1/C1 was correlated with alcohol drinking and unsafe drinking water. MTHFR and CYP4502E1 analysis of gene-gene interaction showed that individuals with the MTHFR677 （C/T ＋ T/T） and CYP4502EIC1/ C1 genotypes had a 7.41-fold （95% CI： 3.60-15.25） risk of developing EC compared with those who carried the MTHFR677C/C and CYP4502E1 RsaI C1/C2 ＋ C2/C2 genes, and the interaction rate was higher than that of the two factors alone. CONCLUSION： Low consumption of green vegetables and fresh fruits, alcohol drinking, and unsafe water （shallow well, or river） and polymorphisms in MTHFR and CYP4502E1 genes are important risk factors for EC. There is a synergistic interaction among polymorphisms in MTHFR and CYP4502E1 genes and environment factors. MTHFR and CYP4502E1 genes can be used as biomarkers for prevention of EC in Kazakh, Xinjiang Uygur Autonomous Region, China.

Mechanisms of Cytochrome C Extraction by Reverse Micelles

The extraction of cytochrome C was carried out by means of phase transfer technique with three different reverse micellar systems, i.e. , a CTAB micellar solution in n butyl alcohol chloroform(volume ratio 4∶1), an AOT micellar solution in isooctane and a SDSS D 2EHPA micellar solution in isooctane. The extraction mechanisms were studied. The results show that the extraction mechanisms for the same proteins with different types of reverse micellar systems can be distinct. The extraction of cytochrome C with CTAB and SDSS D 2EHPA reverse micellar systems are carried out according to the mechanism of electrostatic interaction. However, in the extraction of cytochrome C with the AOT reverse micellar system, the electrostatic interaction between the protein and the surfactant is not important.

Structure of Cytochromec and Its Platinum-modified Derivatives by Fourier Transform Infrared Spectroscopy

The secondary structures of native cytochrome c(cyt c) in both solid and solution states and four platinum modified cyt c derivatives in solution were determined by means of Fourier transform infrared spectroscopy. It was found that the secondary structure of cyt c in solid state is similar to that in the solution. In the cases of platinum modified cyt c derivatives, when the binding sites of platinum complex are on or near the surface of the protein, its secondary structure is similar to that of native cyt c. However, when the platinum complex binds to Met 80 ligand and causes the replacement of the second axial ligand by non native Lys 79 ligand or H 2O supplied by solvent, there is a significant difference between the structures of low or high spin state cyt c derivatives and that of native cyt c. The results suggest that axial ligand Met 80 residue plays an important role in stabilizing the secondary structure of cyt c.

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.

Label-free surface-enhanced infrared spectro-electro-chemical analysis of the Redox potential shift of cytochrome c complexed with a cardiolipin-containing lipid membrane of varied composition

In this study, a lipid membrane was fabricated by fusing cardiolipin-phosphatidylcholine（CL_PC, 1：4） vesicles onto a hydrophobic surface of 1-dodecanethiol（DT） preadsorbed on a nanostructured gold film. By changing the concentration of the DT adsorption solution, we constructed a series of CL PC-DT bilayers with different hydrophobicity to study the effects of lipid membrane characteristics on the adsorption conformation of cytochrome c（Cyt c）. Electrochemical analysis showed that the formal potential is 0.24 V for Cyt c-CL_PC-DT（10）, 0.2 V for Cyt c-CL_PC-DT（20）, and 0.16 V for Cyt c-CL_PC-DT（40） — a gradual positive shift with the decreasing DT concentration — relative to the potential of native cyt c（0.02 V）. Potential-induced surface-enhanced infrared adsorption difference spectroscopy revealed that the gradual positive shift of the formal potential of CL-bound cyt c is determined by the environment with the gradually lowered dielectric constant for the heme cofactor in CL-bound cyt c（Fe^3＋）.

OSW-1 Induced Apoptosis in Hepatocellular Carcinoma through Generation of ROS, Cytochrome C and Noxa Activation Independent of p53 with Non-Activation of Caspase-3

Aim: To study the antitumor mechanism of OSW-1 in hepatocellular carcinoma. Materials and Methods: The expression profiling microarray was carried out to extract RNA from SK-Hep-1 which suffered from OSW-1. ρ0-SK-Hep-1 was maintained SK-Hep-1 in MEM containing 100 μg/L ethidium bromide (EB), 1 mM sodium pyruvate and 50 μg/ml uridine for 40 days. Then confirmed COX-I and COX-II of mitochondrial DNA were knocked out. Cells suffered from OSW-1 or doxorubicin. Then cells were washed twice with cold PBS and incubated with DCFH-DA. Fluorescent signal was recorded by using Infinite 200 Pro multimode Plate readers. Results: OSW-1 elevates generation of ROS and Cytochrome C which are associated with the induction of apoptosis in SK-Hep-1 cells. We also demonstrate that OSW-1 does not depend on p53 to up-regulate the BH3-only protein Noxa. What is more noteworthy that the Caspase-9 and FADD are down-regulated in above process. Conclusion: OSW-1 induced special apoptosis is different from the mitochondrial death pathway and the death receptor pathway and final result is not Caspase family’s activating. This provides a novel theory that nonmalignant cells are significantly less sensitive to OSW-1 than cancer cell lines.

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.

Effects on the Early Development of the Pearl Sac by Cytochrome c,Yolk Lecithin and Polyvinyplyrro

Mantle pieces of Hyriopisis cumingii was treated by three kinds of solution,These mantle pieces were inserted together with paraffin nucleuses into the connective tissue of three groups of Hyriopisis cumingii.These operated animals were cultured in a pool for a month.Several pearl sacs were put out and immersed by Bouin's solution after every five days.Sections of each animals were made by histological method.Those without treatment were in a control group.Observations of these sections showed that cytochrome c and yolk lecithin have an accelerated roles on pearl sac development and pearl layer secretion.No effects on pearl sac development by polyvinyplyrrolidone(PVP).

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.

Cytochrome-c aptamer functionalized Pt nanoclusters for enhanced chemodynamic therapy

Catalysis-based chemodynamic therapy(CDT)is an emerging cancer treatment strategy which uses a Fenton-like reaction to kill tumor cells by catalyzing endogenous hydrogen peroxide(H_(2)O_(2))into a toxic hydroxyl radical(·OH).The performance of CDT is greatly dependent on PDT agent.Herein,mitochondria-targeting Pt nanoclusters were synthesized using cytochrome c aptamer(CytcApt)as template.The obtained CytcApt-PtNCs can produce.OH by H_(2)O_(2)under the acidic conditions.Moreover,CytcApt-PtNCs could kill 4T1 tumor cells in a pH-dependent manner,but had no side effect on normal 293T cells.Therefore,CytcApt-PtNCs possess excellent therapeutic effect and good biosafety,indicating their great potential for CDT.

Study on the Structural Effect of Maltoligosaccharides on Cytochrome c Complexes Stabilities by Native Mass Spectrometry

Noncovalent interactions between ligands and targeting proteins are essential for understanding molecular mechanisms of proteins.In this work,we investigated the interaction of Cytochrome c(Cyt c)with maltoligosaccharides,namely maltose(Mal Ⅱ),maltotriose(Mal Ⅲ),maltotetraose(Mal Ⅳ),maltopentaose(Mal Ⅴ),maltohexaose(Mal Ⅵ)and maltoheptaose(Mal Ⅶ).Using electrospray ionization mass spetrometry(ESI-MS)assay,the 1:1 and 1:2 complexes formed by Cyt c with maltoligosaccharide ligand were observed.The corresponding association constants were calculated according to the deconvoluted spectra.The order of the relative binding affinities of the selected oligosaccharides with Cyt c were as MalⅢ>MalⅣ>MalⅡ>MalⅤ>MalⅥ>MalⅦ.The results indicated that the stability of noncovalent protein complexes was intimately correlated to the molecular structure of bound ligand.The relevant functional groups that could form H-bonds,electrostatic or hydrophobic forces with protein’s amino residues played an important role for the stability of protein complexes.In addition,the steric structure of ligand was also critical for an appropriate interaction with the binding pocket of proteins.

Involvement of mitochondrial swelling in cytochrome c release from mitochondria treated with calcium and Alloxan

An early event in the induction of apoptosis is cytochrome c (Cyt c) release from mitochondria. We investigated the involvement of mitochonrial permeability transition (MPT) and mitochondrial swelling in Cyt c release from mitochondria treated with alloxan and/or calcium (Ca2+). When mitochondria were treated with a high concentration of Ca2+. alone or Ca2+. with alloxan (alloxan-Ca2+), the MPT was accompanied by mitochondrial swelling and the release of Cyt c. Cyclosporin A prevented the induction of MPT but only slightly decreased the release of Cyt c. High molecular weight polyethylene glycol almost completely inhibited MPT-dependent osmotic mitochondrial swelling and Cyt c release. However, MPT-independent mitochondrial swelling and Cyt c release induced by exogenous K+. were inhibited by the high molecular weight polyethylene glycol. Ruthenium red strongly decreased the amount of Cyt c released. These results suggest that mitochondrial swelling but not MPT is necessary for Cyt c release induced by Ca2+ alone or alloxan and Ca2+.

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.

Hippocampal mitochondrial cytochrome C oxidase activity and gene expression in a rat model of chronic cerebral ischemia

The present study established a rat model of chronic cerebral ischemia using bilateral common carotid artery permanent ligation to analyze cytochrome C oxidase activity and mRNA expression in hippocampal mitochondria. Results showed significantly decreased cytochrome C oxidase activity and cytochrome C oxidase II mRNA expression with prolonged ischemia time. Further analysis revealed five mitochondrial cytochrome C oxidase II gene mutations, two newly generated mutations, and four absent mutational sites at 1 month after cerebral ischemia, as well as three mitochondrial cytochrome C oxidase III gene mutations, including two newly generating mutations, and one disappeared mutational site at 1 month after cerebral ischemia. Results demonstrated that decreased cytochrome C oxidase gene expression and mutations, as well as decreased cytochrome C oxidase activity, resulting in energy dysmetabolism, which has been shown to be involved in the DatholoQical Process of ischemic brain iniurv.

Direct Electrochemistry of Cytochrome C on the Glassy Carbon Electrode Modified with 1-Pyrenebutyric Acid/MWNTs

With 1-Pymnebutyric acid （PBA） and multiwalled carbon nanotubes （MWNTs）, glassy carbon electrode modified was successfully prepared. In phosphate buffer solution （pH 7,0）, the direct electrochemistry of cytochrome C （Cyt C） was realized. In the cyclic voltammetry experiment two pairs of redox peaks ofCyt C were observed at 0.018 V and -0.314 V （vs. SCE）, respectively. The redox reaction at 0.018 V was diffusion controlled, while the redox reaction at -0.314 V was adsorption controlled.

Electrochemical Probe of Hydroxylation of 4-Nitro-phenol by Cytochrome C with Hydrogen Peroxide

BU Fengquan(Norman Bethune University of Medical Science, Changchun, 130023)(Received Dec. 2, 1996) The reaction of hydrogen peroxide with cytochrome c makes them coupled to lead to the hydroxylation of 4-nitrophenol. In situ electrochemical probe was used to detect the hydroxylation of 4-nitrophenol, which can avoid the tedious extraction procedure, the loss of the active species and the interference of some colored substances in the detection of 4-nitrocatechol by spectroscopic method. The hydroxyl radical scavengers mannitol and sodium benzoate did not eliminate hydroxylation, but the inhibitory effect of uric acid on the hydroxylation lead to the formation of the ferryl species of the protein during the reaction. These studies suggest that the electrochemical probe might efficiently detect the trace 4-nitrocatechol from the onset of the hydroxylation reaction and thus provides a more sensitive tool.