A novel secreted protein FgHrip1 from Fusarium graminearum triggers immune responses in plants

Fusarium graminearum,the primary pathogenic fungus responsible for Fusarium head blight(FHB)in wheat,secretes abundant chemical compounds that interact with host plants.In this study,a secreted protein FgHrip1,isolated from the culture filtrate of F.graminearum,was found to induce typical cell death in tobacco.The FgHrip1 gene was then cloned and expressed in Escherichia coli.Further bioassay analysis showed that the recombinant FgHrip1 induced early defense induction events,such as reactive oxygen species(ROS)production,callose deposition,and up-regulation of defense-related genes in tobacco.Furthermore,FgHrip1 significantly enhanced immunity in tobacco seedlings against Pseudomonas syringae pv.tabaci 6605(Pst.6605)and tobacco mosaic virus(TMV).FgHrip1-treated wheat spikes also exhibited defense-related transcript accumulation and developed immunity against FHB infection.Whereas the expression of FgHrip1 was induced during the infection process,the deletion of the gene impaired the virulence of F.graminearum.Our results suggest that FgHrip1triggers immunity and induces disease resistance in tobacco and wheat,thereby providing new insight into strategy for biocontrol of FHB.

Secreted Frizzled-Related Protein 5 Mediates Wnt5a Expression in Microcystin-Leucine-Arginine-Induced Liver Lipid Metabolism Disorder in Mice

Objective Microcystin-leucine-arginine(MC-LR)exposure induces lipid metabolism disorders in the liver.Secreted frizzled-related protein 5(SFRP5)is a natural antagonist of winglesstype MMTV integration site family,member 5A(Wnt5a)and an anti-inflammatory adipocytokine.In this study,we aimed to investigate whether MC-LR can induce lipid metabolism disorders in hepatocytes and whether SFRP5,which has anti-inflammatory effects,can alleviate the effects of hepatic lipid metabolism by inhibiting the Wnt5a/Jun N-terminal kinase(JNK)pathway.Methods We exposed mice to MC-LR in vivo to induce liver lipid metabolism disorders.Subsequently,mouse hepatocytes that overexpressed SFRP5 or did not express SFRP5 were exposed to MC-LR,and the effects of SFRP5 overexpression on inflammation and Wnt5a/JNK activation by MC-LR were observed.Results MC-LR exposure induced liver lipid metabolism disorders in mice and significantly decreased SFRP5 mRNA and protein levels in a concentration-dependent manner.SFRP5 overexpression in AML12cells suppressed MC-LR-induced inflammation.Overexpression of SFRP5 also inhibited Wnt5a and phosphorylation of JNK.Conclusion MC-LR can induce lipid metabolism disorders in mice,and SFRP5 can attenuate lipid metabolism disorders in the mouse liver by inhibiting Wnt5a/JNK signaling.

Roles of sulfonylurea receptor 1 and multidrug resistance protein 1 in modulating insulin secretion in human insulinoma

BACKGROUND:Sulfonylurea receptor 1(SUR1)and multidrug resistance protein 1(MRP1)are two prominent members of multidrug resistance proteins associated with insulin secretion. The aims of this study were to investigate their expression in insulinomas and their sole and synergistic effects in modulating abnormal insulin secretion. METHODS:Fasting glucose,insulin and C-peptide were measured in 11 insulinoma patients and 11 healthy controls. Prolonged oral glucose tolerance tests were performed in 6 insulinoma patients.Insulin content,SUR1 and MRP1 were detected in 11 insulinoma patients by immunohistochemistry. SUR1 and MRP1 were also detected in 6 insulinoma patients by immunofluorescence. RESULTS:Insulinoma patients presented the typical demons-trations of Whipple’s triad.Fasting glucose of each insulinoma patient was lower than 2.8 mmol/L,and simultaneous insulin and C-peptide were increased in insulinoma patients. Prolonged oral glucose tolerance tests showed that insulin secretion in insulinoma patients were also stimulated by high glucose.Immunohistochemistry and immunofluorescence staining showed that SUR1 increased,but MRP1 decreased in insulinoma compared with the adjacent islets. CONCLUSIONS:The hypersecretion of insulin in insulinomas might be,at least partially,due to the enrichment of SUR1. In contrast,MRP1,which is down-regulated in insulinomas, might reflect a negative feedback in insulin secretion.

Mannogalactoglucan from mushrooms protects pancreatic islets via restoring UPR and promotes insulin secretion in TIDM mice

Type 1 diabetes mellitus(T1DM) lacks insulin secretion due to autoimmune deficiency of pancreaticβ-cells.Protecting pancreatic islets and enhancing insulin secretion has been therapeutic approaches.Mannogalactoglucan is the main type of polysaccharide from natural mushroom,which has potential medicinal prospects.Nevertheless,the antidiabetic property of mannogalactoglucan in T1DM has not been fully elucidated.In this study,we obtained the neutral fraction of alkali-soluble Armillaria mellea polysaccharide(AAMP-N) with the structure of mannogalactoglucan from the fruiting body of A.mellea and investigated the potential therapeutic value of AAMP-N in T1DM.We demonstrated that AAMP-N lowered blood glucose and improved diabetes symptoms in T1DM mice.AAMP-N activated unfolded protein response(UPR) signaling pathway to maintain ER protein folding homeostasis and promote insulin secretion in vivo.Besides that,AAMP-N promoted insulin synthesis via upregulating the expression of transcription factors,increased Ca^(2+) signals to stimulate intracellular insulin secretory vesicle transport via activating calcium/calmodulin-dependent kinase Ⅱ(CamkⅡ) and cAMP/PKA signals,and enhanced insulin secretory vesicle fusion with the plasma membrane via vesicle-associated membrane protein 2(VAMP2).Collectively,these studies demonstrated that the therapeutic potential of AAMP-N on pancreatic islets function,indicating that mannogalactoglucan could be natural nutraceutical used for the treatment of T1DM.

Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

Hypermethylation and expression regulation of secreted frizzled-related protein genes in colorectal tumor

AIM： To investigate the functions of promoter hypermethylation of secreted frizzled-related proteins （sFRPs） genes in colorectal tumorigenesis and progression. METHODS： The promoter hypermethylation and expression of sFRP genes in 72 sporadic colorectal carcinomas, 33 adenomas, 18 aberrant crypt foci （ACF） and colorectal cancer cell lines RKO, HCT116 and SW480 were detected by methylation-specific PCR and reverse transcription PCR, respectively. RESULTS： None of the normal colorectal mucosa tissues showed methylated bands of any of four sFRP genes, sFRP1, 2, 4 and 5 were frequently methylated in colorectal carcinoma, adenoma and ACF （sFRP1 〉 85%, sFRP2 〉75%, sFRP5 〉 50%）, and the differences between three colorectal tissues were not significant （P 〉 0.05）. IVlethylation in colorectal tumors was more frequent than in normal mucosa and adjacent normal mucosa. The mRNA of sFRP1-5 genes was expressed in all normal colorectal mucosa samples. Expression of sFRP1, 2, 4 and 5 and sFRP1, 2 and 5 was downregulated in carcinoma and adenoma, respectively. The downregulation of sFRP2, 4 and 5 was more frequent in carcinoma than in adenoma. Expression of sFRP3 which promoter has no CpG island was downregulated in only a few of colorectal tumor samples （7/105）. The downregulation ofsFRP1, 2, 4 and 5 expression was significantly associated with promoter hypermethylation in colorectal tumor. After cells were treated by DAC/TSA combination, the silenced sFRP mRNA expression could be effectively re-expressed in colorectal cancer cell lines. CONCLUSION： Hypermethylation of sFRP genes is a common early event in the evolution of colorectal tumor, occurring frequently in ACF, which is regarded as the earliest lesion of multistage colorectal carcinogenesis. It appears to functionally silence sFRP genes expression. Methylation of sFRP1, 2 and 5 genes might serve as indicators for colorectal tumor.

Secreted Frizzled-Related Protein 5 (SFRP5) in Patients with Obstructive Sleep Apnea

Objective Obstructive sleep apnea (OSA) is closely related to obesity, insulin resistance and inflammation. Secreted frizzled-related protein 5 (SFRP5) is a recently discovered adipokine. It is involved in insulin resistance and inflammation in obesity. This study aimed at evaluating the association between SFRP5and sleeping characteristics as well as biochemical parameters of OSA patients.Methods This was a prospective case control study. Nondiabetic OSA patients and controls were consecutively recruited and divided into three groups: OSA group, apnea–hypopnea Index (AHI)≥5/h; healthy controls with normal body mass index (BMI); obese controls without OSA, and BMI > 24.0 kg/m2. All participants underwent polysomnography (PSG). Plasma SFRP5 was examined using enzyme-linked immunosorbent assay (ELISA). Blood biochemical examinations, including fasting blood glucose (FBG), lipid profile, hypersensitive Creactive protein (hsCRP), were performed early in the morning after PSG. Patients with severe OSA were treated with nasal continuous positive airway pressure (nCPAP), and plasma SFRP5 was repeatedly measured for comparison.Results Sixty-eight subjects were enrolled in the study, including 38 patients of OSA, whose medium AHI was 58.70 /h (36.63, 71.15), 20 obese controls, and 10 healthy controls. The plasma SFRP5 level of OSA patients was not significantly different from that of healthy controls or obese controls. In OSA patients, SFRP5 level correlated positively with triglyceride level (r=0.447, P=0.005) and negatively with LDL-cholesterol level and HDLcholesterol level (r=?0.472 and P=0.003; r=?0.478 and P=0.002; respectively). SFRP5 level was not found correlating with FBG, AHI, or any of nocturnal hypoxia parameters. After overnight nCPAP treatment, plasma SFRP5 levels of OSA patients did not change significantly (t=1.557, P = 0.148) compared to that of pretreatment.Conclusions In nondiabetic OSA patients, plasma SFRP5 is associated with the lipid profile. However,no correlation was observed between SFRP5 and FBG or sleep parameters. The SFRP5 level of OSA patients did not differ from that of non-OSA individuals in our study.

Protein induced by vitamin K absence or antagonist-Ⅱ versus alpha-fetoprotein in the diagnosis of hepatocellular carcinoma: A systematic review with meta-analysis

Background: As a promising biomarker of hepatocellular carcinoma(HCC), protein induced by vitamin K absence or antagonist-Ⅱ(PIVKA-Ⅱ) has been studied extensively. However, its diagnostic capability varies across HCC studies. This study aimed to compare the performance of PIVKA-Ⅱ with alpha-fetoprotein(AFP) in the diagnosis of HCC. Data sources: A systematic literature search was conducted to identify the studies from MEDLINE, Embase and Cochrane Library Databases, which were published up to December 20, 2017 to compare the diagnostic capability of PIVKA-Ⅱ and AFP for HCC. The data were pooled using random effects model. Pooled sensitivity and specificity were calculated. Summary receiver operating characteristic curve(ROC) was employed to evaluate the diagnostic accuracy of each marker. Results: Thirty-one studies were included. The pooled sensitivity(95% CI) of PIVKA-Ⅱ and AFP was 0.66(0.65–0.68) and 0.66(0.65–0.67), respectively in diagnosis of HCC; and the corresponding pooled specificity(95% CI) was 0.89(0.88–0.90) and 0.84(0.83–0.85), respectively. The area under the ROC curve(AUC) of PIVKA-Ⅱ and AFP was 0.856(0.817–0.895) and 0.770(0.728–0.811), respectively. Subgroup analysis showed that PIVKA-Ⅱ was superior to AFP in terms of the AUC for both small HCC( < 3 cm) [0.863(0.825–0.901) vs 0.717(0.658–0.776)] and large HCC( ≥ 3 cm) [0.854(0.811–0.897) vs 0.729(0.682–0.776)]; for American [0.926(0.897–0.955) vs 0.698(0.594–0.662)], European [0.772(0.743–0.801) vs 0.628(0.594–0.662)], Asian [0.838(0.812–0.864) vs 0.785(0.764–0.806)] and African [0.812(0.794–0.840) vs 0.721(0.675–0.767)] HCC patients; and for HBV-related [0.909(0.866–0.951) vs 0.714(0.673–0.755)] and mixed-etiology [0.847(0.821–0.873) vs 0.794(0.772–0.816)] HCC. Conclusion: This meta-analysis indicates that PIVKA-Ⅱ is better than AFP in terms of the accuracy for diagnosing HCC, regardless of tumor size, patient ethnic group, or HCC etiology.

Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer

AIM： To identify the methylation of secreted frizzled-related protein 1 （SFRP1） in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients. METHODS： We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific （MSP） PCR and RT-PCR respectively. Fisher＇s exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1. RESULTS： In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2′-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 （44%） and 8 （15%） specimens respectively （x^2= 10.34, P 〈 0.01）. Loss of SFRP1 expression was detected in 17（33%） and 6 （12%） specimens respectively （x^2= 6.75, P 〈 0.01）. There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type. CONCLUSION： SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.

Computational Analysis of Signal Peptide-Dependent Secreted Proteins in Saccharomyces cerevisiae

Computer based software such as the SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.

Suppression of Breast Cancer Proliferation and Induction of Apoptosis via AKT and ERK1/2 Signal Transduction Pathways by Synthetic Polypeptide Derived from Viral Macrophage Inflammatory Protein Ⅱ

SDF-1α,a ligand for the chemokine receptor CXCR4,is well known for mediating the migration of breast cancer cells.In a previous study we demonstrated that a synthetic 21-mer peptide antagonist of CXCR4（NT21MP） derived from the viral macrophage inflammatory protein Ⅱ could antagonize tumor growth in vivo by inhibiting cellular proliferation and inducing apoptosis in breast cancer cells.However,the role of SDF-1α in the signaling pathways underlying the proliferation of human breast cancer cells and associated signaling pathways and inhibiting signal pathways of NT21MP remained unclear.The present study investigated the mechanism of NT21MP on anti-tumor in breast cancer in vitro.The effect of NT21MP on the viability of cells was determined by the MTT assay.Annexin V-FITC and PI staining was performed to detect early stage apoptosisin SKBR3 cells treated with SDF-1α and AMD3100 or NT21MP.Western blotting techniques were used to assay the composition of phosphoproteomics and total proteins present in the SKBR3 breast cancer cells.RT-PCR and Western blotting technique were used to detect the effect of NT21MP and AMD3100 on Bcl-2 and Bax expression.The results indicated that SDF-1α prevented apoptosis and promoted the proliferation of SKBR3 human breast cancer cells.As compared with untreated SKBR3 cells,Treatment with SDF-1α significantly increased cell viability,and NT21MP abolished the protective effects of SDF-1α dose-dependently（P0.05）.There was a significant decrease in the percentage of apoptotic cells after SDF-1α treatment as compared with control group（2.7%±0.2% vs.5.7%±0.4%,P0.05）.But pretreatment of SKBR3 cells with NT21MP significantly attenuated the antiapoptotic effects of SDF-1α as compared with SKBR3 cells without NT21MP pretreatment.The proliferative and anti-apoptotic effects of SDF-1α in SKBR3 cells were associated with an increase in AKT and ERK1/2 phosphorylation as well as a decrease in Bax expression and an increase in Bcl-2 expression.These changes in intracellular processes were blocked by NT21MP in a dose-dependent manner（P0.05）.In conclusion,NT21MP efficiently inhibits SDF-1α-induced proliferation and antiapoptosis in SKBR3 cells by reducing the levels of phosphorylated AKT and ERK1/2,as well as decreasing the ratio of expression of Bcl-2 relative to Bax.

Effect of glycosides of Cistanche on the expression of mitochondrial precursor protein and keratin type Ⅱ cytoskeletal 6A in a rat model of vascular dementia

Glycosides of Cistanche（GC）is a preparation used extensively for its neuroprotective effect against neurological diseases,but its mechanisms of action remains incompletely understood.Here,we established a bilateral common carotid artery occlusion model of vascular dementia in rats and injected the model rats with a suspension of GC（10 mg/kg/day,intraperitoneally）for 14 consecutive days.Immunohistochemistry showed that GC significantly reduced p-tau and amyloid beta（Aβ）immunoreactivity in the hippocampus of the model rats.Proteomic analysis demonstrated upregulation of mitochondrial precursor protein and downregulation of keratin type II cytoskeletal6A after GC treatment compared with model rats that had received saline.Western blot assay confirmed these findings.Our results suggest that the neuroprotective effect of GC in vascular dementia occurs via the promotion of neuronal cytoskeleton regeneration.

Aberrant methylation of secreted protein acidic and rich in cysteine gene and its significance in gastric cancer

BACKGROUND Aberrant methylation in DNA regulatory regions could downregulate tumor suppressor genes without changing the sequences.However,our knowledge of secreted protein acidic and rich in cysteine(SPARC)and its aberrant methylation in gastric cancer(GC)is still inadequate.In the present research,we performed fundamental research to clarify the precise function of methylation on SPARC and its significance in GC.AIM To investigate promoter methylation and the effects of the SPARC gene in GC cells and tissues and to evaluate its clinical significance.METHODS Plasmids that overexpressed the SPARC gene were transfected into human GC BGC-823 cells;non-transfected cells were used as a control group(NC group).Quantitative real-time polymerase chain reaction and western blotting(WB)were then used to detect the expression of SPARC.Methylation-specific polymerase chain reaction was executed to analyze the gene promoter methylation status.Cell viability was measured by the cell counting kit-8 assay.The migration and invasion ability of cells were detected by scratch assays and transwell chamber assays,respectively.Cell cycle events and apoptosis were observed with a flow cytometer.RESULTS The expression of SPARC mRNA in GC tissues and cells was significantly lower and showed differing degrees of hypermethylation,respectively,than that in normal adjacent tissues and control cells.Treatment with 5-Aza-2’-deoxycytidine(5-Aza-Cdr)was able to restore the expression of SPARC and reverse promoter hypermethylation.Overexpression of the SPARC gene significantly inhibited proliferation,migration,and invasion of GC cells,while also causing cell cycle arrest and apoptosis;the NC group exhibited the opposite effects.CONCLUSION This study demonstrated that SPARC could function as a tumor suppressor and might be silenced by promoter hypermethylation.Furthermore,in GC cells,SPARC inhibited migration,invasion,and proliferation,caused cell cycle arrest at the G0/G1 phase,and promoted apoptosis.

Protein induced by vitamin K absence or antagonist Ⅱ-producing gastric cancer

Protein induced by vitamin K absence or antagonist Ⅱ(PIVKA-Ⅱ) is a putative specific marker of hepatocellular carcinoma(HCC),but it may also be produced by asmall number of gastric cancers.To date,16 cases of PIVKA-Ⅱ-producing gastric cancer have been reported,2 of which were reported by us and all of which were identified in Japan.There are no symptoms specific to PIVKA-Ⅱ-producing gastric cancer,and the representative clinical symptoms are general fatigue,appetite loss,and upper abdominal pain.Serum alpha-feto-protein(AFP)levels are also increased in almost allcases.Liver metastasis is observed in approximately 80% of cases and portal vein tumor thrombus is ob-served in approximately 20% of cases.Differential diagnosis between metastatic liver tumor and HCC is often difficult.Grossly,almost all cases appear as advanced gastric cancer.Histologically,a hepatoid pattern is observed in many cases,in addition to a moderately to poorly differentiated adenocarcinoma component.The production of PIVKA-Ⅱ and AFP is usually confirmed using immunohistochemical staining.Treatment and prognosis largely depends on the existence of liver meta-stasis,and the prognosis of patients with liver metas-tasis is very poor.PIVKA-Ⅱ may be produced during the hepatocellular metaplasia of the tumor cells.

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels

Malignant melanoma, characterized by invasive local growth and early formation of metastases, is the most aggressive type of skin cancer. Melanoma inhibitory activity （MIA）, secreted by malignant melanoma cells, interacts with the cell adhesion receptors, integrins a4131 and 05131, facilitating cell detachment and promoting formation of me- tastases. In the present study, we demonstrate that MIA secretion is confined to the rear end of migrating cells, while in non-migrating cells MIA accumulates in the actin cortex. MIA protein takes a conventional secretory pathway including coat protein complex I （COPI）- and coat protein complex II （COPII）-dependent protein transport to the cell periphery, where its final release depends on intracellular Ca2＋ ions. Interestingly, the Ca2＋-activated K＋-channel, subfamily N, member 4 （KCa3.1）, known to be active at the rear end of migrating cells, was found to support MIA secretion. Secretion was diminished by the specific KCa3.1 channel inhibitor TRAM-34 and by expression of dominant- negative mutants of the channel. In summary, we have elucidated the migration-associated transport of MIA protein to the cell rear and also disclosed a new mechanism by which KCa3.1 potassium channels promote cell migration.

Effects of Calmodulin-dependent Protein Kinase Ⅱ Inhibitor,KN-93,on Electrophysiological Features of Rabbit Hypertrophic Cardiac Myocytes

Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.

An Improved Approach for Rapidly Identifying Different Types of Gram-Negative Bacterial Secreted Proteins

Protein secretion plays an important role in bacterial lifestyles. In Gram-negative bacteria, a wide range of proteins are secreted to modulate the interactions of bacteria with their environments and other bacteria via various secretion systems. These proteins are essential for the virulence of bacteria, so it is crucial to study them for the pathogenesis of diseases and the development of drugs. Using amino acid composition (AAC), position-specific scoring matrix (PSSM) and N-terminal signal peptides, two different substitution models are firstly constructed to transform protein sequences into numerical vectors. Then, based on support vector machine (SVM) and the “one to one”?algorithm, a hybrid multi-classifier named SecretP v.2.2 is proposed to rapidly and accurately?distinguish different types of Gram-negative?bacterial secreted proteins. When performed on the same test set for a comparison with other methods, SecretP v.2.2 gets the highest total sensitivity of 93.60%. A public independent dataset is used to further test the power of SecretP v.2.2 for predicting NCSPs, it also yields satisfactory results.

Isolation and Characterization of Proteins Interacting with Activin Type Ⅱ Receptors

Regulation of the number of aetivin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, interacting with activin type II receptors, was searched and identified by using yeast two-hybrid screening. ARIPzip is a splicing variant of ARIP2. This has been discussed previously. ARIPzip can specifically interact with ActR Ⅱ A, and is widely distributed in mouse tissues. Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIV2 can decrease these activities. These data suggest that the C-terminal rezions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.

MicroRNA-219 alleviates glutamate-induced neurotoxicity in cultured hippocampal neurons by targeting calmodulin-dependent protein kinase Ⅱ gamma

Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.

Taurolithocholate impairs bile canalicular motility and canalicular bile secretion in isolated rat hepatocyte couplets

AIM: To investigate the effects of taurolithocholate (TLC)on the canalicular motility in isolated rat hepatocyte cou-plets (IRHC).METHODS: TLC was added to IRHC at concentrationsof 10 and 50 μmol/L, respectively. In each group, fi vetime-lapse movies containing 3 representative bile cana-liculi were taken under phase-contrast microscopy for12 h. The number of bile canalicular contractions andthe intervals between consecutive canalicular contrac-tions were calculated. Furthermore, the effects of TLC onIRHC were examined by transmission electron micros-copy.RESULTS: The bile canalicular contractions were spon-taneous and forceful in the controls. Active vesicularmovement was observed in the pericanalicular region.Immediately after the addition of TLC, the bile canaliculiwere deformed, and canalicular bile was incorporatedinto the vacuoles. The canaliculi were gradually dilated,and canalicular contractions were markedly inhibited byTLC. The vesicular movements became extremely slowin the pericanalicular region. The number of canalicularcontractions significantly decreased in the TLC-treatedgroups, as compared with that in the controls. The timeintervals were prolonged, as the TLC dosage increased,indicating that bile secretion into the canaliculi wasimpaired with TLC. Transmission electron microscopyrevealed the lamellar transformation of the canalicularmembranes in IRHC treated with TLC.CONCLUSION: TLC impairs both the bile canalicularcontractions and the canalicular bile secretion, possiblyby acting directly on the canalicular membranes in TLC-induced cholestasis.