Cyt C Research Articles

The hooded mutant of Lathyrus odoratus (Fabaceae) is associated with a cycloidea gene mutation

The hooded (hdd) floral mutant of Lathyrus odoratus L. (sweet pea) has a concave standard petal compared with the flat standard petal of the wild type. This trait was used by Bateson, Punnett, and Saunders in early studies of Mendelian inheritance (c.1905). Here we provide four lines of evidence that this phenotype results from a mutation in the CYCLOIDEA2 (CYC2) gene. (i) CYC2 is expressed in the standard petals of wild-type L. odoratus, whereas the same methods fail to detect expression in hdd plants. (ii) Genomic sequencing reveals that the CYC2 gene sequence of hdd plants is truncated at the TCP box and likely nonfunctional. (iii) In a population of 118 plants, the hdd phenotype cosegregated with the mutant allele of CYC2 without exception. (iv) CYC2 is known to act as a dorsal petal identity gene. Consistent with this, the standard petal in hdd flowers has the epidermal and pigment characteristics of wing petals, indicating that the hdd mutation results in a shift in dorsiventral petal-type identity. We conclude that the mutation in CYC2 is responsible for the hdd phenotype, and is therefore the L. odoratus equivalent of the lobed standard (lst1) mutant in Pisum.

Predicting DNA hybridization kinetics from sequence.

Hybridization is a key molecular process in biology and biotechnology, but to date there is no predictive model for accurately determining hybridization rate constants based on sequence information. Here we report a weighted neighbor voting (WNV) prediction algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants. To construct this algorithm we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (36nt subsequences of the CYCS and VEGF genes) at temperatures ranging from 28°C to 55°C. Automated feature selection and weighting optimization resulted in a final 6-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 3 with ≈91% accuracy, based on leave-one-out cross-validation. Accurate prediction of hybridization kinetics allows design of efficient probe sequences for genomics research.

Effect of methylene blue preconditioning on ischemia-reperfusion injury in isolated rat lungs

Objective To evaluate the effect of methylene blue(MB)preconditioning on ischemia-reperfusion(I/R)injury in isolated rat lungs. Methods Eighteen pathogen-free healthy male Sprague-Dawley rats, aged 3 months, weighing 240-320 g, were divided into 3 groups(n=6 each)using a random number table: sham operation group(group Sham), lung I/R group(group I/R)and methylene blue preconditioning group(group MB). A model of isolated lung I/R injury was established in pentobarbital sodium-anesthetized rats.MB 2 mg/kg was intraperitoneally injected at 2 h before stopping perfusion in group MB.Isolated lungs were perfused for 20 min, followed by 45-min ischemia, and then reperfused for 60 min in I/R and MB groups.At 60 min of reperfusion, the activity of lactic dehydrogenase(LDH)in the perfusate was detected, wet weight(W)and dry weight(D)was determined, W/D ratio was calculated, and the levels of malondialdehyde(MDA), ATP, reactive oxygen species(ROS)and superoxide dismutase(SOD)were measured in lung tissues.Mitochondria and cytoplasm were isolated from lung tissues for determination of mitochondrial membrane potential(MMP), degree of mitochondrial swelling and content of cytochrome C(Cyt c)in cytoplasm.Apoptotic cells in lung tissues were detected using TUNEL, and apoptotic index was calculated. Results Compared with group Sham, the activity of LDH in perfusate, W/D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly increased, the degree of mitochondrial swelling was aggravated, and the content of ATP and MMP were decreased in I/R and MB groups, and the SOD activity was significantly decreased in group I/R(P<0.05). Compared with group I/R, the activity of LDH in perfusate, W/D ratio, levels of ROS, MDA and Cyt c in cytoplasm and apoptosis index were significantly decreased, the degree of mitochondrial swelling was attenuated, and the activity of SOD, content of ATP and MMP were increased in group MB(P<0.05). Conclusion Methylene blue preconditioning can reduce I/R injury in isolated rat lungs, and the mechanism may be related to improving mitochondrial function and inhibiting cell apoptosis. Key words: Methylene blue; Ischemic preconditioning; Reperfusion injury; Lung

Usnic acid uses mitochondrial apopitotic pathway in it’s antitumoral role

Objective : Usnic acid is a secondary metabolite in lichens whose role has not been completely elucidated. Lichen extracts containing usnic acid have been utilized in medicine, perfumery, cosmetics, and ecology. Usnic asit inhibits cell growth, induces the cell cycle arrest and apoptosis in human lung carcinoma. Methods :Herein we analyzed the antitumoral effect of usnic acid on the same cell line. We analyzed the gene expression results on APOPT1, CYCS, APAF1, CASP3/9, TNF , BCL2, BCL2L1 and AIFM1 genes which have possible role on cell apopitosis. Results : Usnic acid has stimulatory effect on APOPT1, CYCS, APAF1, CASP3, CASP9 gene expressions in our experiments. Conclusion : Usnic acid has antitumoral effect on cancer cells, affecting on mithocondrial apopitotic genes.

Changes and clinical significances of mitochondrial coupling factor 6 and cytochrome C in neonatal sepsis

Objective To evaluate the levels of plasma coupling factor 6(CF6) and cytochrome C(Cyt-c) in neonatal sepsis, and to explore the clinical significance in neonatal sepsis. Methods A total of 88 neonates admitted to Hunan Children′s Hospital from January 2015 to April 2015 were collected.Neonates were divided into non-sepsis group(n=42) and sepsis group(n=46). According to the severity of infection, the non-sepsis group was further divided into non-infection group(n=20) and common infection group(n=22); the sepsis group was further divided into general sepsis group (31 cases, no organ failure) and severe sepsis group (15 cases, combined with multiple organ failure). Femoral venous blood was collected in all patients before the use of antibiotics after admission.The levels of Cyt-c and CF6 in plasma were measured by ELISA, and the levels of C-reactive protein(CRP), procalcitonin (PCT) were measured.The changes of CF6 and Cyt-c between these groups were compared, and the sensitivity and specificity with the traditional sepsis index (CRP, PCT) were analyzed.The correlation between the levels of CF6, Cyt-c and neonatal critical illness score was analyzed. Results (1)In sepsis group, the levels of CF6 and Cyt-c[(109.7±8.9)pg/ml and (44.5±4.9)ng/ml] were significantly higher than those in the non-sepsis group[(46.3±6.0)pg/ml, (31.8±6.7)ng/ml, P<0.01, respectively]. (2) In the non-infection group, common infection group, general sepsis group and severe sepsis group, the levels of CF6 were (32.1±8.9)pg/ml, (59.3±7.2)pg/ml, (79.3±5.9)pg/ml, and (172.6±6.1)pg/ml, respectively; the levels of Cyt-c were (29.3±8.6)ng/ml, (35.4±4.1) ng/ml, (43.1±5.9) ng/ml, and (44.5±5.9)ng/ml, respectively.The differences between these groups were significant(P<0.01). (3)The receiver operating characteristic curve showed that the sensitivity and specificity of CF6 were 0.761, 0.732, and the Cyt-c were 0.739, 0.714.(4)Cyt-C and CF6 were negatively correlated with the neonatal critical illness score(r=-0.599, P<0.001; r=-0.337, P<0.01). Conclusion The levels of CF6 and Cyt-c increase in neonatal sepsis.The damage of mitochondria may be one of the pathological mechanisms in neonatal sepsis.The levels of CF6 and Cyt-c were closely related to the severity of neonatal sepsis. Key words: Sepsis; Mitochondria; Mitochondrial coupling factor 6; Cytochrome C,

Effect of exogenous hydrogen sulfide on mitochondrion-dependent apoptosis in lung tissues of rats with endotoxemia

Objective To evaluate the effect of exogenous hydrogen sulfide (H2S) on mitochondrion-dependent apoptosis in lung tissues of rats with endotoxemia. Methods Forty healthy male Sprague-Dawley rats, aged 2-3 months, weighing 250-310 g, were divided into 5 groups (n=8 each) using a random number table: control group (group C), lipopolysaccharide (LPS) group, low dose sodium hydrosulphide (NaHS) group (group L-NaHS), moderate dose NaHS group (group M-NaHS) and high dose NaHS group (group H-NaHS). Endotoxemia was induced by IV LPS 5 mg/kg in chloral hydrate-anesthetized rats.The equal volume of 0.9% sodium chloride solution was intravenously injected in group C. NaHS (an exogenous donor of H2S) 0.78, 1.56 and 3.12 mg/kg were intraperitoneally injected at 3 h after LPS injection in L-NaHS, M-NaHS and H-NaHS groups, respectively.The rats were sacrificed at 6 h after injection of LPS or 0.9% sodium chloride solution, and lungs were removed for examination of the mitochondrial ultrastructure of lung tissues and for determination of apoptosis in lung cells (by flow cytometry) and expression of caspase-3, caspase-9, Bcl-2 and Bax protein and mRNA in lung tissues (by Western blot or real-time polymerase chain reaction). The apoptosis rate and ratio of Bcl-2 expression to Bax expression (Bcl-2/Bax ratio) were calculated.The expression of cytochrome c (Cyt c) in cytoplasm and mitochondria of lung tissues was detected by Western blot. Results The apoptosis rate was significantly increased, the expression of caspase-3, caspase-9, Bcl-2 and Bax protein and mRNA was up-regulated, Bcl-2/Bax ratio was decreased, the expression of Cyt c in cytoplasm was up-regulated, and the expression of Cyt c in mitochondria was down-regulated in group LPS (P 0.05), and the damage to mitochondrial ultrastructure was significantly mitigated in M-NaHS and H-NaHS groups. Conclusion The mechanism by which exogenous H2S inhibits cell apoptosis in lung tissues may be related to inhibition of mitochondrion-dependent apoptosis in rats with endotoxemia. Key words: Hydrogen sulfide; Endotoxemia; Lung; Apoptosis; Mitochondria

Novel Pelagic Iron-Oxidizing Zetaproteobacteria from the Chesapeake Bay Oxic-Anoxic Transition Zone.

Chemolithotrophic iron-oxidizing bacteria (FeOB) could theoretically inhabit any environment where Fe(II) and O2 (or nitrate) coexist. Until recently, marine Fe-oxidizing Zetaproteobacteria had primarily been observed in benthic and subsurface settings, but not redox-stratified water columns. This may be due to the challenges that a pelagic lifestyle would pose for Zetaproteobacteria, given low Fe(II) concentrations in modern marine waters and the possibility that Fe oxyhydroxide biominerals could cause cells to sink. However, we recently cultivated Zetaproteobacteria from the Chesapeake Bay oxic–anoxic transition zone, suggesting that they can survive and contribute to biogeochemical cycling in a stratified estuary. Here we describe the isolation, characterization, and genomes of two new species, Mariprofundus aestuarium CP-5 and Mariprofundus ferrinatatus CP-8, which are the first Zetaproteobacteria isolates from a pelagic environment. We looked for adaptations enabling strains CP-5 and CP-8 to overcome the challenges of living in a low Fe redoxcline with frequent O2 fluctuations due to tidal mixing. We found that the CP strains produce distinctive dreadlock-like Fe oxyhydroxide structures that are easily shed, which would help cells maintain suspension in the water column. These oxides are by-products of Fe(II) oxidation, likely catalyzed by the putative Fe(II) oxidase encoded by the cyc2 gene, present in both CP-5 and CP-8 genomes; the consistent presence of cyc2 in all microaerophilic FeOB and other FeOB genomes supports its putative role in Fe(II) oxidation. The CP strains also have two gene clusters associated with biofilm formation (Wsp system and the Widespread Colonization Island) that are absent or rare in other Zetaproteobacteria. We propose that biofilm formation enables the CP strains to attach to FeS particles and form flocs, an advantageous strategy for scavenging Fe(II) and developing low [O2] microenvironments within more oxygenated waters. However, the CP strains appear to be adapted to somewhat higher concentrations of O2, as indicated by the presence of genes encoding aa3-type cytochrome c oxidases, but not the cbb3-type found in all other Zetaproteobacteria isolate genomes. Overall, our results reveal adaptations for life in a physically dynamic, low Fe(II) water column, suggesting that niche-specific strategies can enable Zetaproteobacteria to live in any environment with Fe(II).

The CYCLOIDEA-RADIALIS module regulates petal shape and pigmentation, leading to bilateral corolla symmetry in Torenia fournieri (Linderniaceae).

The diverse pigmentation patterns of flower corollas probably result from pollinator-mediated selection. Previous studies demonstrated that R2R3-MYB factors may have been recruited in the regulation of corolla pigmentation. However, how R2R3-MYBs became so diverse in their regulation of different pigmentation patterns remains unclear. Here, we studied a Lamiales species, Torenia fournieri, which has elaborate zygomorphic flowers with dorsal-ventral asymmetries in corolla pigmentation. We found recent gene duplication events in CYCLOIDEA-like (CYC-like) and RADIALIS-like (RAD-like) genes, and functionally analyzed three dorsal-specific expression factors: TfCYC1, TfCYC2, and TfRAD1. We found that the CYC-RAD module coordinates petal shape and corolla pigmentation, as ectopic expression of TfCYC2 or TfRAD1 disrupted the asymmetric corolla pigmentation pattern and produced strongly dorsalized flowers. Dorsal petal identity was lost when TfCYC2 was down-regulated or when TfRAD1 was knocked out. In T.fournieri, the diversified CYC and RAD genes have evolved regulatory loops, and TfCYC2 binds directly to the regulatory regions of an R2R3-MYB factor gene, TfMYB1, which might lead to its asymmetric expression and ultimately establish the asymmetric pigmentation pattern. These findings support the existence of a regulatory module that integrates dorsal-ventral patterning and asymmetric corolla pigmentation in T.fournieri.

Nano Copper Induces Apoptosis in PK-15 Cells via a Mitochondria-Mediated Pathway.

Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40-100μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40μg/mL. To summarize, nano copper significantly (P<0.05) decreased the level of SOD and increased the level of MDA in PK-15 cells. Altogether, these results suggest that nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.

Evolution and Expression Patterns of CYC/TB1 Genes in Anacyclus: Phylogenetic Insights for Floral Symmetry Genes in Asteraceae.

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development.

Apoptosis and autophagy of retinal ganglion cell-5 cells induced by hydrostatic pressure

Background Researches showed that the mitochondrial-mediated apoptosis and autophagy play an important role in the survival of retinal ganglion cells (RGCs) in glaucoma.However, whether high pressure will lead to mitochondrial-mediated apoptosis and autophagy is not elucidated. Objective This study was conducted to explore whether elevated pressure can directly induce the mitochondrial-mediated apoptosis and autophagy in cultured RGC-5 cells in vitro. Methods RGC-5 cells were exposed to self-made pressure device and treated by 0, 20, 40 and 60 mmHg pressure for four hours in pressurized bottles.RGC-5 cells were incubated simultaneously in sealed incubator bottles and served as normal control.The morphological changes of the cells were examined under the inverted phase-contrast microscope.The apoptosis percentages of the cells were detected by flow cytometry.Mitochondrial membrane potentials of the cells were assessed using the JC-1, a fluorescent dye.And the expressions of cytochrome C (Cyt-c), microtubule associated protein light chain 3 (LC3) and Beclin-1 protein in the cells was detected by Western blot. Results Cultured cells showed fusiform shape in the normal control group and 0 mmHg group with more dendritic process.The cell density was obviously reduced and the number of dead cells was increased in the 20, 40 and 60 mmHg groups.The apoptotic percentage was (15.69±0.77)%, (15.77±1.14)%, (18.30±1.07)%, (23.28±1.33)% and (34.47±1.17)% in the normal control group and 0, 20, 40 and 60 mmHg groups, respectively, showing a significant intergroup difference (F=150.90, P<0.001), and the apoptotic percentage in the 40 mmHg group and 60 mmHg group was significantly increased in comparison with the normal control group (both at P<0.01). The mitochondrial membrane potential was high in the normal control group and 0 mmHg group, with the reddish fluorescence in the cells, and the reddish fluorescence was weakened in the 20 mmHg group and 40 mmHg group.The lowing of mitochondrial membrane potential was seen in the 60 mmHg group, with the green fluorescence.Compared with the normal control group, the expression of Cyt-c, LC3-Ⅱ and Beclin-1 proteins in pressured groups was correspondingly increased (all at P<0.05). Conclusions Elevated pressure induces the morphologic change of RGC-5 cells, results in mitochondrial membrane potential reduction and mitochondrial-mediated apoptosis and autophagy. Key words: Glaucoma/physiopathology; Pressure; Mitochondria/metabolism; Retinal ganglion cells/metabolism; Apoptosis; Autophagy

Role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation

Objective To evaluate the role of autophagy in hydrogen-induced inhibition of apoptosis in hippocampal neurons in a rat model of orthotopic liver transplantation (OLT). Methods Fifty-six pathogen-free healthy adult male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were used in the study.Thirty-two rats were selected and assigned into 4 groups (n=8 each)using a random number table: sham operation group (group S), OLT group, hydrogen-rich saline group (group HS) and chloroquine group (group CQ). The other 24 rats severed as the donors.In group S, laparotomy was performed, and the related blood vessels were isolated.The model of OLT was established in OLT, HS and CQ groups.In group OLT, normal saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group HS, hydrogen-rich saline 6 ml/kg was slowly injected via the inferior vena cava at 5 min before anhepatic phase.In group CQ, autophagy inhibitor chloroquine 60 mg/kg was injected intraperitoneally at 1 h before establishment of the model, and the other treatments were similar to those previously described in group HS.At 6 h of reperfusion, the rats were sacrificed and hippocampi were isolated for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, for pathological examination (with light microscope), and for detection of cell apoptosis (by TUNEL staining) and expression of autophagy- and apoptosis-related proteins caspase-3, cytochrome c (Cyt c), microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ), Beclin-1 and p53 in hippocampal tissues (by Western blot analysis). Apoptosis index (AI) was calculated. Results Compared with group S, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3, Cyt c, LC3Ⅱ, Beclin-1 and p53 was up-regulated in OLT, HS and CQ groups (P<0.05). Compared with group OLT, the MDA content and AI were significantly decreased, the SOD activity was increased, the expression of caspase-3 and Cyt c was down-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was up-regulated in group HS (P<0.05). Compared with group HS, the MDA content and AI were significantly increased, the SOD activity was decreased, and the expression of caspase-3 and Cyt c was up-regulated, and the expression of LC3Ⅱ, Beclin-1 and p53 was down-regulated in group CQ (P<0.05). Conclusion The mechanism by which hydrogen inhibits apoptosis in hippocampal neurons is related to promotion of autophagy in a rat model of OLT. Key words: Autophagy; Hydrogen; Liver transplantation; Brain injury; Apoptosis

Effect of dietary zinc on morphological characteristics and apoptosis related gene expression in the small intestine of Bama miniature pigs

To investigate the effects of dietary zinc (Zn) on small intestinal mucosal epithelium, 6-month-old male Bama miniature pigs were randomly allocated into three groups and treated with three levels of Zn (Control, T1, and T2 diet supplemented with 0, 50, and 1500mg/kg Zn, respectively, as zinc sulfate) for 38days. The samples of small intestine tissues, serum, and feces were collected. The results showed that Zn concentrations of small intestine in the T2 group were higher than those in the control and T1 groups (p<0.05). In the T2 group, the pharmacological dose of dietary Zn treatment caused marked damage to the small intestinal epithelium. The expression of Bax, cleaved caspase-3, and caspase-8 were increased in the duodenum and the jejunum of the T2 group (p<0.05). The mRNA transcript levels of BAX, CYCS and CASP3 genes were upregulated in the duodenum and the jejunum of the T2 group. We concluded that a diet with a pharmacological dose of Zn increased the accumulation of Zn and the expression of Bax, cleaved caspase-3, and caspase-8, which might activate the apoptosis and lead to the marked injury of porcine small intestinal epithelium.

Molecular Analysis of Chinese Celastrus and Tripterygium and Implications in Medicinal and Pharmacological Studies.

Celastrus and Tripterygium species, which are used in traditional Chinese medicine, have attracted much attention due to their anti-tumor promoting and neuroprotective activities, in addition to their applications in autoimmune disorders. However, systematic relationships between them and among species are unclear, and it may disturb their further medicinal utilization. In the present study, the molecular analysis of combined chloroplast and nuclear markers of all Chinese Celastrus and Tripterygium was performed, and clear inter- and intra-genus relationships were presented. The result suggests that Tripterygium constitute a natural monophyletic clade within Celastrus with strong support value. Fruit and seed type are better than inflorescence in subgeneric classification. Chinese Celastrus are classified for three sections: Sect. Sempervirentes (Maxim.) CY Cheng & TC Kao, Sect. Lunatus XY Mu & ZX Zhang, sect. nov., and Sect. Ellipticus XY Mu & ZX Zhang, sect. nov. The phylogenetic data was consistent with their chemical components reported previously. Owing to the close relationship, several evergreen Celastrus species are recommended for chemical and pharmacological studies. Our results also provide reference for molecular identification of Chinese Celastrus and Tripterygium.

Pyrroloquinoline quinone inhibited oxidative stress induced-apoptosis of Schwann cells via mitochondrial apoptotic signaling pathway in vitro

To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs). SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group,H2O2 induced group,H2O2 + PQQ treated group.CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis. In this study, the S-100 positive cells were more than 95％,cell proliferation was decreased in H2O2 induced SCs,apoptotic rate was increased, mitochondrial transmnembrane potential was decreased,CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased. PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.

Effect of sevoflurane postconditioning on expression of Pim-1 kinase during myocardial ischemia-reperfusion in rats: an in vitro experiment

Objective To evaluate the effect of sevoflurane postconditioning on the expression of Pim-1 kinase during myocardial ischemia-reperfusion(I/R)in rats. Methods Male Sprague-Dawley rats, weighing 250-300 g, were used in this study.After the animals were anesthetized, their hearts were immediately removed and retrogradely perfused with an oxygenated K-H solution at 37 ℃ in a Langendorff apparatus.Thirty-six isolated rat hearts were assigned into 3 groups(n=12 each)using a random number table: control group(group C), group I/R and sevoflurane postconditioning group(group SP). The hearts were subjected to ischemia for 30 min followed by 120 min of reperfusion to establish the model of myocardial I/R injury.In group SP, the hearts were perfused with K-H solution saturated with 3% sevoflurane for 15 min starting from the beginning of reperfusion.Heart rate(HR), left ventricular end-diastolic pressure(LVEDP), left ventricular developed pressure(LVDP)and the maximum rate of increase and decrease of left ventricular pressure(±dp/dtmax) were recorded at the end of equilibration and 30, 60, 90 and 120 min of reperfusion.Myocardial tissues were obtained at T2 for determination of the expression of Pim-1 kinase, Bcl-2 and cytochrome c(Cyt c)in cytoplasm and mitochondria by Western blot.The hearts were selected at T4 for determination of myocardial infarct size(by TTC staining)and for examination of mitochondrial ultrastructure(with transmission electron microscope). Results Compared with group C, HR, LVDP and ±dp/dtmax were significantly decreased, and LVEDP was increased at T1-4, the myocardial infarct size was enlarged, the expression of Pim-1 and Bcl-2 in cytoplasm and mitochondria was down-regulated, the expression of Cyt c in cytoplasm was up-regulated, and the expression of Cyt c in mitochondria was down-regulated in group I/R(P<0.05). Compared with group I/R, HR, LVDP and ±dp/dtmax were significantly increased, and LVEDP was decreased at T1-4, the myocardial infarct size was decreased, the expression of Pim-1 kinase and Bcl-2 in cytoplasm and mitochondria was up-regulated, the expression of Cyt c in cytoplasm was down-regulated, and the expression of Cyt c in mitochondria was up-regulated in group SP(P<0.05). The damage to mitochondria was significantly attenuated in group SP as compared with group I/R, and was aggravated as compared with group C. Conclusion The mechanism by which sevoflurane postconditioning attenuates myocardial I/R may be related to up-regulation of Pim-1 kinase expression in rats. Key words: Proto-oncogene proteins; Anesthetics, inhalation; Ischemic postconditioning; Myocardial reperfusion injury

Effect of hypothermia on expression of dynamin-related protein 1 in brain tissues during global cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of hypothermia on the expression of dynamin-related protein 1(Drp1)in brain tissues during global cerebral ischemia-reperfusion(I/R)in rats. Methods Thirty-six healthy male Sprague-Dawley rats, weighing 280-320 g, were divided into 3 groups(n=12 each)using a random number table: sham operation group(group Sham), global cerebral I/R group(group I/R)and hypothermia group(group H). Cardiac arrest was induced by transoesophageal cardiac pacing followed by cardiopulmonary resuscitation to establish the global cerebral I/R model in anesthetized rats in I/R and H groups.In group H, the body temperature(rectal temperature)was cooled down to 32-34 ℃ within 15 min starting from the beginning of reperfusion, and maintained at this level for 6 h. At 72 h of reperfusion, neurological deficit was scored, and the rats were sacrificed, and the whole brain was removed for examination of the pathological changes in hippocampal CA1 region and for determination of normal pyramidal cell count and neuronal apoptosis in hippocampal CA1 region and expression of Drp1 and cytochrome c(Cyt c)in hippocampal tissues(by Western blot). The apoptosis rate was calculated. Results Compared with group S, the neurological deficit score and apoptosis rate were significantly increased, and the number of normal pyramidal cells was decreased in I/R and H groups, the expression of Drp1 and Cyt c in hippocampal tissues was significantly up-regulated in group I/R(P 0.05). Compared with group I/R, the neurological deficit score and apoptosis rate were significantly decreased, the number of normal pyramidal cells was increased, and the expression of Drp1 and Cyt c in hippocampal tissues was down-regulated in group H(P<0.05). Conclusion The mechanism by which hypothermia inhibits cell apoptosis during global cerebral I/R may be related to down-regulation of Drp1 expression in rats. Key words: Hypothermia, induced; Reperfusion injury; Brain; Dynamins

Clozapine modifies the differentiation program of human adipocytes inducing browning.

Administration of second-generation antipsychotic drugs (SGAs) often leads to weight gain and consequent cardio-metabolic side effects. We observed that clozapine but not six other antipsychotic drugs reprogrammed the gene expression pattern of differentiating human adipocytes ex vivo, leading to an elevated expression of the browning marker gene UCP1, more and smaller lipid droplets and more mitochondrial DNA than in the untreated white adipocytes. Laser scanning cytometry showed that up to 40% of the differentiating single primary and Simpson–Golabi–Behmel syndrome (SGBS) adipocytes had the characteristic morphological features of browning cells. Furthermore, clozapine significantly upregulated ELOVL3, CIDEA, CYC1, PGC1A and TBX1 genes but not ZIC1 suggesting induction of the beige-like and not the classical brown phenotype. When we tested whether browning induced by clozapine can be explained by its known pharmacological effect of antagonizing serotonin (5HT) receptors, it was found that browning cells expressed 5HT receptors 2A, 1D, 7 and the upregulation of browning markers was diminished in the presence of exogenous 5HT. Undifferentiated progenitors or completely differentiated beige or white adipocytes did not respond to clozapine administration. The clozapine-induced beige cells displayed increased basal and oligomycin-inhibited (proton leak) oxygen consumption, but these cells showed a lower response to cAMP stimulus as compared with control beige adipocytes indicating that they are less capable to respond to natural thermogenic anti-obesity cues. Our data altogether suggest that novel pharmacological stimulation of these masked beige adipocytes can be a future therapeutic target for the treatment of SGA-induced weight gain.

Genome-Wide Identification, Characterization and Expression Analysis of the TCP Gene Family in Prunus mume.

TCP proteins, belonging to a plant-specific transcription factors family, are known to have great functions in plant development, especially flower and leaf development. However, there is little information about this gene family in Prunus mume, which is widely cultivated in China as an ornamental and fruit tree. Here a genome-wide analysis of TCP genes was performed to explore their evolution in P. mume. Nineteen PmTCPs were identified and three of them contained putative miR319 target sites. Phylogenetic and comprehensive bioinformatics analyses of these genes revealed that different types of TCP genes had undergone different evolutionary processes and the genes in the same clade had similar chromosomal location, gene structure, and conserved domains. Expression analysis of these PmTCPs indicated that there were diverse expression patterns among different clades. Most TCP genes were predominantly expressed in flower, leaf, and stem, and showed high expression levels in the different stages of flower bud differentiation, especially in petal formation stage and gametophyte development. Genes in TCP-P subfamily had main roles in both flower development and gametophyte development. The CIN genes in double petal cultivars might have key roles in the formation of petal, while they were correlated with gametophyte development in the single petal cultivar. The CYC/TB1 type genes were highly detected in the formation of petal and pistil. The less-complex flower types of P. mume might result from the fact that there were only two CYC type genes present in P. mume and a lack of CYC2 genes to control the identity of flower types. These results lay the foundation for further study on the functions of TCP genes during flower development.

Effect of propofol on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation injury

Objective To evaluate the effect of propofol on mitochondrial fission in a rat hippocampal neuron model of hypoxia/reoxygenation (H/R) injury. Methods Primarily cultured hippocampal neurons of neonatal Sprague-Dawley rats were randomly divided into 4 groups (n= 42 each) using a random number table: control group (group C); vehicle group (group V); H/R group; H/R+ propofol group (group H/R+ P). In group V, H/R was not produced, the vehicle dimethyl sulfoxide with the final concentration of 0.01% was added, and the cells were then incubated for 6 h. In group H/R, the hippocampal neurons were subjected to oxygen-glucose deprivation for 6 h followed by 20 h reoxygenation.In group H/R+ P, propofol with the final concentration of 1 μmol/L was added at 6 h of hypoxia.At 20 h of reoxygenation, the cell apoptosis (using flow cytometry), Ca2+ concentrations in cytoplasm (with the laser scanning confocal microscope), calcineurin (CaN) activities (by enzyme-linked immunosorbent assay), and expression of mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission 1 (Fis1), and apoptosis-related proteins cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) (by Western blot) were measured.The apoptotic rate was calculated. Results Compared with group C, the apoptotic rate, Ca2+ concentrations, CaN activities, and expression of Drp1, Fis1, Cyt c and AIF were significantly increased in H/R and H/R+ P groups (P 0.05). Compared with group H/R, the apoptotic rate, Ca2+ concentrations, CaN activities, and expression of Drp1, Fis1, Cyt c and AIF were significantly decreased in group H/R+ P (P<0.05=. Conclusion Propofol can reduce the H/R injury to rat hippocampal neurons through inhibiting mitochondrial fission. Key words: Propofol; Anoxia; Mitochondria; Hippocampus; Neurons