Effects of epinephrine on angiogenesis-related gene expressions in cultured rat cardiomyocytes

Epinephrine is often used for the treatment of patients with heart failure, low cardiac output and cardiac arrest. It can acutely improve hemodynamic parameters; however, it does not seem to improve longer term clinical outcomes. Therefore, we hypothesized that epinephrine may induce unfavorable changes in gene expression of cardiomyocyte. Thus, we investigated effects of epinephrine exposure on the mediation or modulation of gene expression of cultured cardiomyocytes at a genome-wide scale. Our investigation revealed that exposure of cardiomyocytes to epinephrine in an in vitro environment can up-regulate the expression ofangiopoietin-2 gene （~ 2.1 times）, and down-regulate the gene expression of neuregulin 1 （-3.7 times）, plasminogen activator inhibitor-1 （-2.4 times） and SPARC-related modular calcium-binding protein-2 （-4.5 times）. These changes suggest that epinephrine exposure may induce inhibition of angiogenesis-related gene expressions in cultured rat cardiomyocytes. The precise clinical significance of these changes in gene expression, which was induced by epinephrine exposure, warrants further experimental and clinical investigations.

Effects of milrinone on inflammatory response-related gene expressions in cultured rat cardiomyocytes

Congestive heart failure(CHF)is defined as a cardiac dysfunction leading to low cardiac output and inadequate tissue perfusion.Intravenous positive inotropes are used to increase myocardial contractility in hospitalized patients with advanced heart failure.Milrinone is a phosphodiesterase Ⅲ inhibitor and used most commonly for inotropic effect.The well-known PROMISE study investigated the effects of milrinone on mortality in patients with severe CHF,and concluded that long-term therapy with milrinone increased morbidity and mortality armong patients with advanced CHF.Previous studies have suggested that phosphodiesterase inhibitors can have potential effects on inflammatory pathways.Hence,we hypothesized that milrinone may alter inflammatory gene expressions in cardiomyocytes,thus leading to adverse clinical outcomes.We used rat cardiomyocyte cell line H9 C2 and studied the impact of exposing cardiomyocytes to milrinone(10 μmol/L)for 24 hours on inflammatory gene expressions.RNA extracted from cultured cardiomyocytes was used for whole rat genome gene expression assay(41 000 genes).The following changes in inflammatory response-related gene expressions were discovered.Genes with increased expressions included:THBS2(+ 9.98),MMP2(+3.47),DDIT3(+2.39),and ADORA3(+3.5).Genes with decreased expressions were:SPP1(-5.28)and CD14(-2.05).We found that the above mentioned gene expression changes seem to indicate that milrinone may hinder the inflammatory process which may potentially lead to adverse clinical outcomes.However,further in vivo and clinical investigations will be needed to illustrate the clinical relevance of these gene expression changes induced by milrinone.

EFFECT OF PHORBOL ESTER ON cAMP－DEPENDENT PROTEIN KINASE ACTIVITY IN CARDIOMYOCYTES

Cardiomyocytes isolated from neonatal rats were treated with phorboll 2-myristatel 3-acetate (PMA ) ranging from 10(-11)to 10-7 mol/L for 20 min, causing cytosol protein kinase A (PKA) activity to decrease while particulate PKA activity increase in a concentration-dependent manner. The change of PKA activity induced by PMA was abolished completely by pretreatment of polymyxin B or depletion of protein kinase C (PKC). Type II PKA activity in particulate fraction was enhanced remarkably, while that of type I PKA was not altered when the cells were treated with 100 nmol/L PMA. The results suggested that subcellular distribution and activity of PKA in cardiomyocytes may be regulated by PKC.

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background:Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period.Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells(hiPS-CM),researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM.In our previous studies,we separately supplemented two polyunsaturated fatty acids(PUFAs),docosahexaenoic acid(DHA)or arachidonic acid(AA),to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation,fatty acid metabolism,and cellular adhesion.The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force.Methods:This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM.In addition to the contractile force and mRNA measurements used in the previous study,we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis,measured glucose and fatty acids metabolites,and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation.Results:It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors,and the supplementations may activate the CD36_ERRγmetabolic pathway.DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters.The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant.AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36.However,finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings.Conclusion:DHA and AA,as having been supplemented in infant formulas,have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually,although they were able to enter the cellular metabolic system.The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM,which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.

Applicability of the P19CL6 cells as a model of cardiomyocytes – a transcriptome analysis

The P19CL6 cell-line, a clone of the P19 mouse embryonal carcinoma cell-line, has been exten-sively used as a model for cardiomyocytes as these cells can be differentiated into a cardio-myocyte phenotype upon incubation with di-methyl sulfoxide. Uniquely, these cells can be observed to “beat” when monitored by mi-croscopy. We started investigating the response of P19CL6 cells to fatty acids, but highly vari-able results lead us to investigate the phenotype of the P19CL6 cells in more depth. In this study we demonstrated that the P19CL6 cells are re-sponsive to adrenaline, but loose the “beating” phenotype after 16 passages in culture. Analysis of specific mRNA transcripts indicated that the P19CL6 cells express both cardiac- and skeletal muscle-specific genes, while global analysis of microarray data showed clear differences be-tween the P19CL6 cells and cardiac tissue of embryonic or adult origin. In conclusion, our observations suggest caution in the use of the P19CL6 cells as a model of cardiomyocytes unless rigorous validation for the intended analysis has been undertaken.

Effects of docosahexaenoic acid or arachidonic acid supplementation on gene expression and contractile force of rat cardiomyocytes in primary culture

While fatty acids play essential roles in the physiology of the myocardium,conventional culture media contain little lipid.We previously revealed that rat neonatal myocardium mainly contains docosahexaenoic(DHA),linoleic(LA),and arachidonic(AA)acids as polyunsaturated fatty acids(PUFAs),and these contents in cultured cardiomyocytes derived from fetal rats were markedly lower than those in the neonatal myocardium.In this study,we first assessed the effects of supplementation of DHA,LA,or AA on the fatty acid contents and the percentage change of contractile area in primarily cultured rat cardiomyocytes.Based on this assessment,we then evaluated the effects of DHA or AA supplementation on mRNA expression and further directly measured the contractile force of cardiomyocytes with the supplementations.This study revealed that percentage change of contractile area was maximized under 20μM DHA or 50μM AA supplementation while LA supplementation did not affect this contraction index,and that a widespread upregulation tendency of the mRNA expression related to differentiation,maturity,fatty acid metabolism,and cell adhesion was seen in the cultured cardiomyocytes with supplementation of DHA or AA.In particular,upregulation of the gene expression of cellular adhesion molecules connexin43 and N-cadherin were remarkable,whereas the effects on differentiation and maturation were less pronounced.Correspondingly,the increase of the percentage change of the contractile area of cardiomyocyte clusters in culture dishes with the supplementations was significant,whereas the enhancement of the contractile force was modest.These results suggest that supplementation of DHA or AA to the fetal cardiomyocyte culture may play effective roles in preventing the de-differentiation of the cardiomyocytes in culture and that the enhancement of the contractile performance may be mainly attributed to the improvement of intercellular connection.

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G_(2)/M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.

Expressions of p16 and FHIT Proteins During Esophageal Carcinomatous Development in High Incidence Area of Esophageal Carcinoma

Objective： To detect the changes of p16 and FHIT and investigate their relationship in esophageal squamous cell carcinoma development by measuring their expression levels in normal squamous epithelium tissue, mild, moderate, severe dysplasia lesions, carcinoma in situ and invasive squamous cell carcinomas. Methods： Expressions of p16 protein and FHIT protein were detected and analyzed in 17 cases of normal squamous epithelium, 16 cases of mild dysplasia, 16 cases of moderate dysplasia, 17 cases of severe dysplasia, 10 cases of carcinoma in situ, and 18 cases of esophageal squamous cell carcinoma by immunohistochemical method. Results： With increasing histopathologic grades, the expressions of pl6 and FHIT became gradually lower. There was no remarkable difference of p16 and FHIT expressions between the normal and mild dysplasia group （P〉0.05）, but the differences between the normal and other groups were all significant （P〈0.05）. There was no remarkable difference among the squamous cell carcinoma group, the moderate and severe dysplasia groups, and the carcinoma in situ group （P〉0.05）, but significant differences existed in the expressions of p16 and FHIT proteins between the squamous cell carcinoma and the normal groups, and between the squamous cell carcinoma and the mild dysplasia groups （P〈0.05）. There was an association of descending trend between p16 and FHIT protein expressions. Conclusion： Reduced expressions of pl6 and/or FHIT proteins possible play an important role in the early occurrence of esophageal cancer. There was a positive correlation between the expressions of p16 and FHIT proteins.

Effect of different therapies of Chinese medicine on the expressions of c-Fos and c-Jun proteins in hippocampus of rats with post-stroke depression

BACKGROUND ： c-fos and c-jun, the important immediate early genes （IEG）, are regarded as the markers for the location and function of neuronal activity, as well as the third signal messengers, they couple the stress stimulation and the gene expression in neuron, and hippocampus is involved in the process of signal transmission after stress stimulation induced depression. OBJECTIVE： To observe the therapeutic effects of Bushen Yiqi （tonifying kidney to benefit qi）, Huoxue Huayu （promoting blood circulation to dissipate blood stasis） and Ditan Kaiqiao （eliminating phlegm for resuscitation） on the expressions of c-Fos and c-Jun proteins in hippocampus and spontaneous behaviors of rats with post-stroke depression （PSD）, and compare the results with those of fluoxetine, which is known to have definite effect on depression. DESIGN： A randomized controlled tna SETTING ： Zhejiang College of Traditional Chinese Medicine MATERIALS ： The trial was completed in Zhejiang College of Traditional Chinese Medicine from January to July in 2003. Fifty-six healthy adult Wistar male rats of clean grade, weighing （250±50） g, were randomly divided into 7 groups with 8 rats in each group： control group, model group, forced swimming group, Bushen Yiqi group; Huoxue Huayu, Ditan Kaiqiao group and fluoxetine group. The Bushen Yiqi Tang contained Renshen, Huangqi, Heshouwu, Gouqi, Shudi, etc., crude drugs 1 800 g/L. The Huoxue Huayu Tang contained Danshen, Chuanxiong, Chishao, Yujin, etc., crude drugs 3 600 g/L. The Ditan Kaiqiao Tang contained Banxia, Danxing, Changpu, Yuanzhi, etc., crude drug 1 000 g/b METHODS： ① Except the control group and forced swimming group, rats in the other groups were made into PSD models by deligating the bilateral common carotid artedes permanently. ② Rats in the control group, model group and forced swimming group were intragastncally perfused by saline （3 mL for each time）; those in the Bushen Yiqi group, Huoxue Huayu, Ditan Kaiqiao group and fluoxetine group were intragastncally perfused with Bushen Yiqi Tang （18 g/kg）, Huoxue Huayu Tang （9 g/kg）, Ditan Kaiqiao Tang （9 g/kg） and fluoxetine （2.5 mg/kg） respectively, once a day. ③ At 55 days after model establishment, rats in the forced swimming group were managed according to the Porsolt＇s method. They were placed in water for 15 minutes, and then taken out and dned, no moving-time within 5 minutes was recorded at drying and 24 hours after drying. ④ Measurement of spontaneous behaviors： Except the forced swimming group, the spontaneous behaviors and activities （including horizontal and vertical movements） of rats were observed with the Open-Field method at 28, 42 and 56 days after administration in the other groups. ⑤ The expressions of c-Fos and coJun proteins in hippocampus were determined with the immunohistochemical method, the relative sectional area ratio and average objective gray value of c-Fos and c-Jun positive cells in hip- pocampus were measured with the computerized image analytical system. MAIN OUTCOME MEASURES： The spontaneous behaviors of rats, the relative sectional area ratio and average objective gray value of c-Fos and c-Jun positive cells in hippocampus were observed. RESULTS： Of the 56 rats, 1 died in the forced swimming group, and finally 55 rats were involved in the analysis of results. ① Results of spontaneous activities： At 28 days, the times of crossing movements were obviously fewer in the model group and fluoxetine group [（69.00±37.01）, （98.11 ±36.68） times/3 minutes] than in the control group [（128.44±16.85） times/3 minutes, P 〈 0.01, 0.05], but those in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group had no obvious differences as compared with those in the control group （P 〉 0.05）. At 42 and 56 days, the times of crossing movements were obviously more in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group [（106.44±31.24）, （117.20±23.95）, （134.80±28.18）, （136.36±40.95） times/3 minutes; （117.33±35.91）, （129.60 ±23.78）, （131.90 ±26.81）, （136.09±28.34） times/3 minutes] than in the model group [（64.00±17.51）, （72.86±20.68） times/3 minutes, P 〈 0.01]. The times of rearing movements had no obvious differences among the groups for the three times （P 〉 0.05）. ② The no moving-time within 5 minutes 24 hours after drying was obviously longer than that at drying in the forced swimming group. ③ The average objective gray values of c-Fos positive cells were not obviously different in the Bushen Yiqi group and Ditan Kaiqiao group from the control group （P 〉 0.05）, but lower in the model group than in the control group （69.84±9.82, 75.78±5.89, P 〈 0.01）, and higher in the forced swimming group than in the control group （85.97±10.99, P 〈 0.01）; all higher in the fluoxetine group, Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group than in the model group （81.27±10.73, 74.04±8.34, 83.29±9.89, 70.14±4.92, P 〈 0.05-0.01）. The average objective gray values of c-Jun positive cells were obviously lower in the Bushen Yiqi group than in the control group （68.11 ±6.89, 79.58±5.86, P 〈 0.01）, but all higher in the other groups than in the control group （84.68±7.15, 81.34 ±8.36, 97.51±10.55, 85.68±9.25, 86.19±10.98, P 〈 0.05-0.01）; Those were obviously higher in the fluoxetine group, Huoxue Quyu group and Ditan Kaiqiao group than in the model group （P 〈 0.05-0.01 ）, lower in the Bushen Yiqi group than in the model group （P 〈 0.05）, all obviously lower in the Bushen Yiqi group, Huoxue Quyu group and Ditan Kaiqiao group than in the fluoxetine group （P 〈 0.01）. The relative sectional area ratios of c-Fos and c-Jun positive cells had no obvious differences among the groups （P 〉 0.05）. CONCLUSION ： The methods of Bushen Yiqi, Huoxue Quyu and Ditan Kaiqiao can effectively treat PSD in rats, and the results were equivalent with those of fluoxetine, the actions of the above-mentioned drugs may correlated with their regulation to c-Fos and c-Jun expressions in hippocampus. PSD animal models can be successfully established by both permanent deligation of bilateral common carotid arteries and forced swimming, and the models induced by the former has similar basic cerebrovascular lesions as human stroke in clinic.

Ginsenoside Rb1 Ameliorates Autophagy of Hypoxia Cardiomyocytes from Neonatal Rats via AMP-Activated Protein Kinase Pathway

Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventricles from 1-to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group(normal level oxygen), hypoxia group(500 μmol/L CoCl2), Gs-Rb1 group(200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group(500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group(500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR)+ 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group(1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cel s were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay and cardiac troponin I(cTnI) levels were detected by enzyme-linked immunosorbent assay(ELISA). AMPK activity was assessed by 2’,7’-dichlorofluorescein diacetate(DCFH-DA) ELISA assay. The protein expressions of Atg4 B, Atg5, Atg6, Atg7, microtubule-associated protein 1 A/1 B-light chain 3(LC3), P62, and active-cathepsin B were measured by Western blot. Results: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes(P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A(P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A(P<0.01) and was not affected by AICAR(P=0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B Ⅱ, the LC3BⅡ/Ⅰ ratio and cathepsin B activity of hypoxia cardiomyocytes(P<0.05), each of these protein levels was significantly enhanced by Ara A(all P<0.01), but was not affected by AICAR(all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes(P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A(P<0.05) and were not affected by AICAR(P=0.871). Conclusion: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.

Elementary study of differential expression of total soluble proteins in different life phases of Porphyra yezoensis

Total soluble proteins of different life stages, filamentous sporophytes cultivated in high temperatures, and blade gametophytes harvested in different seasons, were identified by SDS-PAGE. The types and amounts of expressed proteins also varied amongst the samples. The fewest soluble proteins were present in filamentous sporophytes. There were more types and amounts of soluble protein in conchospores than in filamentous sporophytes, but fewer than in bulgy sporophytes. More types of protein were detected in filamentous sporophytes cultivated in high temperatures than in those growing in normal situations. The most types and amounts of protein were found in blade gametophytes in all samples. Blade gametophytes harvested last year and stored at -20 ℃ showed only minor differences in expression of proteins when compared with those harvested in different seasons.

Separation and Identification of Differentially Expressed Proteins in Pistillate Flowers between Different Mulberry Cultivars

[Objective] This study aimed to explore the proteins related to pistillate flower development in different mulberry cultivars. [Method] The total proteins of the pistillate flowers of two mulberry cultivars Dal0 （Morus atropurpurea Roxb.） and SG01 （Morus muIticaulis Perr.） were extracted, separated and detected through two- dimensional electrophoresis （2-DE） and mass spectrometry. [Result] There was sig- nificant difference in the expression of proteins from the pistillate flowers of different mulberry cultivars. From the 2-DE images of Dal0 and SG01, 445_＋17 and 425_＋12 protein spots were respectively detected. The expression levels of 75 protein spots differed significantly. Thirteen spots those were expressed at high levels and well separated were analyzed by mass spectrometry, and nine of them were identified successfully. The nine proteins are involved in the glycometabolism, protein and amino acid metabolism and defense responses during the development of mulberry pistillate flower after they were pollinated. [Conclusion] The findings will provide reference for further study on the molecular mechanism of mulberry pistillate flower de- velopment.

Dissociation of FK506 Binding Protein 12.6 from Ryanodine Receptor Type 2 Is Regulated by cADPR but not β-Adrenergic Stimulation in Mouse Cardiomyocytes

AIMS: β-adrenergic augmentation of Ca2+ sparks and cardiac contractility has been functionally linked to phosphorylation-dependent dissociation of FK506 binding protein 12.

Presence of Fas and Bcl-2 proteins in BEL-7404 human hepatoma cells

AIM To study the expression of Fas and Bcl 2 proteins in BEL 7404 human hepatoma cells in order to analyze the possible relationship between cell growth regulation by alpha fetoprotein(AFP) and Fas/Bcl 2 proteins. METHODS BEL 7404 human hepatoma cells were maintained in RPMI 1640 medium supplemented with 10% new born calf serum. Cells adhered to coverslips were used to detect Fas and Bcl 2 protein expression by the avidin biotin complex (ABC) immunocytochemical assay. RESULTS Immunocytochemical study showed that essentially all the BEL 7404 human hepatoma cells could express Fas and Bcl 2 proteins, although in various amounts. No positive staining for Fas and Bcl 2 proteins was observed when cells were incubated with non relevant sera to establish the specificity. CONCLUSION Fas apoptosis signals and Bcl 2 rescue/survival signals from apoptosis are expressed in BEL 7404 human hepatoma cells. The finding strongly implys that AFP mediated cell apoptosis and growth enhancement are potentially associated with Fas and Bcl 2 proteins present in those cells.

Strategies for production of active eukaryotic proteins in bacterial expression system

Bacteria have long been the favorite expression system for recombinant protein production.However,the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias,protein folding,phosphorylation,glycosylation,mRNA stability and promoter strength.Factors are cited and the methods to convert to soluble and active proteins are described,for example a tiglit control of Escherichia coli milieu,refolding from inclusion body and through fusion technology.

Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta(Busck)

Insect pheromone-binding proteins （PBPs） play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors （ORs）. However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12：Ac and Z8-12：Ac, as well as weaker binding afifnities to Z8-12：OH and 12：OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth.

Protection of Ultra-filtration Extract from Danggui Buxue Decoction(当归补血汤) on Oxidative Damage in Cardiomyocytes of Neonatal Rats and Its Mechanism

Objective： To investigate whether the administration of the ultra-filtration extract from Danggui Buxue Decoction （当归补血汤, EDBD） was able to protect cardiomyocytes from oxidative injury of rats induced by hydrogen peroxide （H2O2） and its potential mechanism. Methods： Myocardial cells from 1- to 2-day-old neonatal rats were cultured in Dulbecco＇s modified Eagle＇s medium low-glucose and Ham＇s F12 medium （1：1）, and the cellular injury was induced by H2O2. The ultra-filtration extract mixture from Angelica sinensis and Hedysarurn po/ybotrys was given in three doses of 3.75, 7.5, and 15 mg/mL. Morphological changes of cardiomyocytes were observed by microscope. Survival rate of myocardial cells was assessed using 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide （Ml-r） assay. The cardiomyocyte damages were estimated by detecting lactate dehydrogenase （LDH） and creatine kinase （CK） releases in the medium, superoxide dismutase （SOD） activities, and intracellular malondialdehyde （MDA） and myeloperoxidase （MPO） contents. The levels of caspase-3 and heat shock protein 70 （hsp70） mRNA expression in cardiomyocytes were measured by reverse transcription polymerase chain reaction. Results： The EDBD could protect the cardiomyocytes from H202 injury in a dose- dependent manner （3.75, 7.50, and 15.00 mg/mL）. The EDBD could significantly decrease LDH and CK leakages and intracellular MDA and MPO contents, increase SOD activity, up-regulate hsp70 expression, and down-regulate caspase-3 expression. Conclusion： The EDBD has protection on cardiomyocytes injured by H202 through improving cell antioxidant ability, up-regulating hsp70 expression, and inhibiting caspase-3 activity.

Beneficial effects of metformin on primary cardiomyocytes via activation of adenosine monophosphate-activated protein kinase

Background Metformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin. Methods Cardiomyocytes were incubated in the presence of 100μmol/L H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide （AICAR） （500μmol/L）, an adenosine monophophate （AMP）-activated protein kinase （AMPK） agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C （an AMPK antagonist, 20μmol/L） for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase （eNOS）, and transforming growth factor （TGF）-β1 were analyzed using immunblotting. Results Metformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor （bFGF）, and tumor necrosis factor （TNF）-α. Conclusions Metformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

Soluble expression of recomb inant cMyc,Klf4,Oct4,and Sox2 proteins in bacteria and transduction into living cells

AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.