EFFECTS OF PHYCOCYANIN ON EXPRESSION OF CytC mRNA AND CASPASE-3 mRNA AFTER FOCAL CEREBRAL ISCHEMIA/REPERFUSION IN RATS

Objective To study the effects of phycocyanin on the expression of Cytochrome C (CytC)genes and Caspase-3 genes after focal cerebral ischemia/reperfusion in rats. Methods A rat middle cerebral ar-tery occlusion (MCAO)/reperfusion model was produced using the intraluminal filament method. The rats were di-vided into three groups: sham operation group, model control group and phycocyanin group. After MCAO, the neu-robehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenyltet-razolium chloride (TTC) staining. The expression of CytC mRNA and Caspase-3 mRNA were determined by in situhybridization. Results In the sham operation group and the model control group, there was only a few CytC-positive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of CytC mRNAbegan 6h after ischemia, reached a maximum at 12h (cortex) -24h (striatum) , then subsided gradually, but stillin high level. In the phycocyanin group, CytC-positive cells were also mainly in cortex and striatum, but the numberof the cells was significantly lower than the number of the model control group. The time-phase pattern of CytCmRNA in the phycocyanin group was similar to the pattern of the model control group. In the sham operation groupand the model control group, there was only a few Caspase-3-positive cells were seen in the normal cerebral tissue.In the model control group, the upregulation of Caspase-3 mRNA began 6h after ischemia, reached a maximum at24h and subsided at 48h, but still in high level. In the phycocyanin group, Caspase-3-positive cells were also mainlyin the penumbral area, but the number of the cells were significantly lower than the number of the model controlgroup. The time-phase pattern of Caspase-3 mRNA in the phycocyanin group was similar to the pattern of the modelcontrol group. Conclusion The over-expression of CytC mRNA and Caspase-3 mRNA might play a key role inischemic cerebral injury after MCAO. Phycocyanin could inhibit the over-expression of CytC mRNA and Caspase-3mRNA in the cerebral cortex, and might play an important role in the protection of ischemic neurons.

Effects of DNA Methylation and Histone Modification on Differentiation-associated Gene Expression in ES,NIH3T3,and NIT-1

The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differentiation-associated genes（Pdx-1,Pax4,MafA and Nkx6.1）,Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR（MeDIP-qPCR） techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification（DNA methylation,H3 acetylation,H3K4m3 and H3K9m3） of these genes and their expression was analyzed.The results showed that：（1） the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; （2） NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells（P〈0.05）; （3） NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells（P〈0.05）,with significantly increased level of gene expression; （4） NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell（P〈0.05）,with no detectable mRNA expression of these genes.It was concluded that histone modification（H3K4m3 and H3K9m3） and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.

EFFECTS OF ANTISENSE OLIGODEOXYNUCLEOTIDES ON EXPRESSION OF CASPASE-3 IN Γ-RADIATION INDUCED APOPTOTIC HL-60 CELLS

Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.

Mitochondrial and nuclear damages and caspase-3 expression in the hippicampal CA3 region of rats with kainic acid induced statuse pilepticus

BACKGROUND: Some scholars believed that the neuronal injury after status epilepticus is apoptosis, the main evidence is the changes of expressions of various apoptosis related genes, such as immediate-early gene, p53 gene and genes of bcl-2 family, etc. But there is still no ultrastructural evidence for apoptosis. OBJECTIVE: To observe the ultrastructural damages of mitochondrion and nucleus and the changes of caspase expression in neurons of hippocampal CA3 region in rats with status epilepticus induced by kainic acid. DESIGN: A randomized controlled study. SETTING: Department of Anesthesiology and Department of Neurology, Qilu Hospital of Shandong University. MATERIALS: Seventy-five adult male Wistar rats of 250-300 g, clean degree, were provided by the experimental animal center of Shandong University. Kainic acid was purchased from Sigma Company (USA); rabbit anti-rat polyclonal antibody caspase-3 from Santa Cruz Company (USA). METHODS: The experiments were carried out in the Department of Anesthesiology, Qilu Hospital of Shandong University from October 2005 to February 2006. ① The 75 rats were randomly divided into experimental group (n =45) and control group (n =30). ② Model establishment, convulsion grading and the judging standards for status epilepticus: Rats in the experimental group were given intraperitoneal injection of kainic acid (10 mg/kg), and those in the control group were injected with saline of the same volume. The time of seizure was recorded and their behavioral manifestations were observed, and the seizure was terminated by intraperitoneal injection of diazepam (10 mg/kg). ③ Observation under electron microscope: At 3, 12 and 24 hours after status epilepticus respectively, bilateral hippocampal tissues were taken out, semithin sections of about 75 nm were prepared after fixation, dehydration and embedding, and then observed under H-800 transmission electron microscope. ④ Immunohistochemical detection: Bilateral hippocampi were removed at 3, 12 and 24 hours after status epilepticus respectively, the fixation, dehydration, transparence, wax immersion and embedding were performed, then serial sections of CA3 region were immunohistochemically determined by the SABC method. Leica QWinV3 image analytical software was applied, then the average number and average gray value of positive cells were calculated. MAIN OUTCOME MEASURES: Results of observation under electron microscope, that of immunohistochemical staining of neurons in hippocampal CA3 region; Comparison of number of caspase-3 positive cells and gray value. RESULTS: All the 75 Wistar rats were involved in the analysis of results. ① Results of observation under electron microscope: At 3 hours after status epilepticus, swelling crista and membranous disintegration were observed under electron microscope. At 24 hours, obvious nuclear changes occurred, and manifested as the side-aggegation of chromatins. ② Results of immunohistochemical detection: In the experimental group, the number of caspase-3 positive cells at 3 hours after status epilepticus had no obvious difference as compared with that in the control group (P > 0.05); At 12 hours, the number and gray value of caspase-3 positive cells in the experimental group were higher than those in the control group (10.49±0.68 vs. 5.33±0.43; 45.57±2.27 vs. 19.79±0.33, P < 0.05), the same results were also observed at 24 hours (37.36±0.57 vs. 5.12±0.47; 115.24±1.22 vs. 18.73±0.42, P < 0.01). CONCLUSION: In the rat models of status epilepticus induced by kainic acid, mitochondrial damage was earlier than the increase of caspase-3 expression and nuclear changes, which suggested that mitochondrion was the key link for the neuronal death after status epilepticus.

Phycocyanin for protecting brain ischemia-reperfusion injury and its effect on the expression of Caspase-3 mRNA

BACKGROUND ; Phycecyanin can anti-oxidize and clear free radial. Whether its protective effect on brain is related to Caspase-3, the promoter and operator of apoptosis, is highly concerned. OBJECTIVE： To observe phycocyanin for protecting nerve function and reducing the size of cerebral infarction of rats with brain ischemia-reperfusion and its effect on the expression of Cespese-3 mRNA. DESIGN ： A randomized controlled experiment. SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： Totally 84 adult healthy female Wistar rats, weighing 210 to 250 g, of clean grade, were provided by the Animal Experimental Center of Shandong University. Phycocyanin （Institute of Oceanography of Chinese Academy of Sciences） was used. METHODS： This experiment was carried out in the Key Laboratory for Prevention and Treatment of Brain Diseases during May to December 2005. ① The rats were randomized into sham-operation group （n=4）, control group （n=-40） and phycocyanin-treated group （n=-40）. Middle cerebral artery occlusion/reperfusion （MACO/R） models were created on the rats of control and phycocyanin-treated groups with suture-occluded method by inserting a thread into left side extemal-internal carotid artery. In the sham-operatien group, inserting suture was omitted. After ischemia for 1 hour and reperfusion for 2 hours, suspension of phycocyanin was intragastdcaUy administrated into the rats of the phycocyanin-treated group at 100 mg/kg , and the same volume of normal saline was isochrenously administrated into the rats of control group as the same. ② Six rats were chosen respectively from the control group and phycocyanin-treated group, then neurologic impairment degrees of rats were evaluated according to Bederson＇s grading. ③ Six rats were chosen respectively from the control and phycocyanin-treated groups. The isolated brain tissue was stained with tdphenyltetrazolium chloride, and then the size of cerebral infarction was calculated with HPIAS-1000 image analytical system by calculating the ratio of cerebral infarction size at each layer and contralateral hemisphere size of the same layer. ④ Twenty--eight rats were chosen respectively from the control and phycocyanin-treated groups, Brain tissue was harvested at reperfusion for 6,12,24 hours and for 2,3,7 and 14 days after ischemia for 1 hour, respectively, 4 rats at each time point. Brain tissue of 4 rats of sham-opera- tion group was harvested at the 24^th hour after operation. Brain tissue sections were performed in situ hybridization detection of Cespase-3 mRNA. MAIN OUTCOME MEASURES： Comparison of neurologic impairment degree, cerebral infarction size and the expression of brain tissue Caspase-3 mRNA of rats between two groups RESULTS： Totally 84 rats entered the stage of result analysis. ① Bederson＇s scores at ischemia and reperfusion for 24 and 48 hours were significantly lower in the phycocyanin-treated group than in the control group（P 〈 0.05）. ② After brain ischemia and reperfusion, the infarction area was the largest in the 3^rc layer in both control and phycocyanin-treated group, which was（25.23±0,47）% and（23.09±120） %, respectively, and the size of infarction area in the 2^nd layer to the 5^th layer was significantly smaller in the phycocyanin-treated group than in the control group （P 〈 0.05）. ③Positive cell counts of brain tissue Caspase-3 mRNA： The number of positive cells of Caspase-3 mRNA of control group was increased from cerebral ischemia and reperfusion 6 hours, reached the peak at ischemia and reperfusion 24 hours, began to decrease 2 days later and positive cells of Caspese-3 mRNA were still expressed on the 14^th day after reperfusion. At ischemia and reperfusion 6,12 and 24 hours as well as 2,3,7 and 14 days, positive cell counts of Caspase-3 at peripheral ischemic area were significantly lower in the phycocyanin-treated greup[（70.67 ±3.65）, （85.06±4.79）, （119.54±5.37）,（74.26±2.19）, （62.06±3.34）, （23.11±1.89）, （10.75±2.63）/visual field] than in the control group [（94.38±8 28）, （108.81 ±16.11）, （140.88±14.47）, （98.13±11.31）, （81.03±9.31）, （31.22±8.86）, （16.06±5.96）Nisual field] （ P 〈 0.05）; and those at central ischemic area were also significantly lower in the phycocyanin-treated group [（33.86±4.01）, （39.51±3.46）, （50.96 ±2.53）, （43.07±4.09）, （36.25 ±3.72）, （9.03±3.87）, （4.91±5.59）/visual field ]than in the control group [（51.35±2.13）, （54.87±3.42）, （61.77±4.94）, （55.69±6.06）, （49.01 ±5.73） ,（12.84±3.37）, （7.32±2.39）/visual field]（P 〈 0.05）. CONCLUSION ： Phycocyanin can obviously improve the neurologic function, reduce the size of brain infarction and down-regulate the expression of Caspase-3 mRNA of rats with ischemia and reperfusion injury, thus protect brain.

Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21^(WAF1) expression and hepatic apoptosis

AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21WAF1gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21WAF1expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21WAF1positive tumors as compared withp21WAF1-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21WAF1,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21WAF1and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21WAF1may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.

Effects of salvianolic acid-A on NIH/3T3 fibroblast proliferation,collagen synthesis and gene expression

AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10-4mol/L-10-7mol/L SA-A for 22 h.The cell viability wasassayed by[3H]proline incorporation,cellproliferation by[3H]TdR incorporation,cellcollagen synthetic rate was measured with[3H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10-4mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10-5mol/L-10-7mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10-6mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10-5mol/L-10-6mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10-5mol/L and10-6mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.

CLINICOPATHOLOGICAL SIGNIFICANCE OF PTEN AND CASPASE-3 EXPRESSIONS IN BREAST CANCER

Objective To investigate the expressions of PTEN and Caspase-3 proteins in human breast carcinoma,and to evaluate their clinicopathological implications during the tumorigenesis and progression of breast cancer.Methods The expressions of PTEN and Caspase-3 proteins in 95 cases of breast cancer and 15 cases of benign breast diseases were investigated immunohistochemically.Correlations between the expression of PTEN protein,Caspase-3 protein,and clinicopathological features of breast cancers were analyzed.Results The loss expression rate of PTEN protein in tumor tissues was significantly higher than that in benign breast diseases(33.7% vs.0,P<0.01).Analysis of the clinicopathological features showed that PTEN expression level was negatively correlated with TNM stage,histological grade,axillary lymph node status,recurrence,and metastasis(P<0.05).The positive expression level of Caspase-3 was negatively correlated with TNM stage(P<0.01),but not related with histological grade,axillary lymph node status,recurrence,or metastasis(P>0.05).In addition,the expression of PTEN protein had significantly positive correlation with the expression of Caspase-3 protein in breast cancer(P<0.01).Conclusion The combination detection of PTEN and Caspase-3 may serve as an important index to estimate the pathobiological behavior and prognosis of breast cancer.

EXPRESSION OF PTEN AND CASPASE-3 AND THEIR CLINICOPATHOLOGICAL SIGNIFICANCE IN PRIMARY GASTRIC MALIGNANT LYMPHOMA

Objective To investigate the expression of PTEN and Caspase-3 in malignant lymphoma of the stomach and explore their role in progression of primary gastric malignant lymphoma. Methods Formalin-fixed paraffin embedded tissues from 56 cases of primary gastric malignant lymphoma and their adjacent non-tumor mucosa were evaluated for PTEN and Caspase-3 protein ex-pression by streptavidin-biotin-complex (SABC) immunohistochemistry. Their expression was compared with clinical tumor parameters with the relationship between PTEN and Caspase-3 expression concerned as well. Results The positive rate of PTEN expression in primary gastric lymphomas(50.0%, 28/56) was significantly lower than that in adjacent non-tumor gastric mucosa(96.4%, 27/28)(P < 0.05). Meanwhile,43 of 56(76.8%)gastric lymphomas indicated Caspase-3 expression, less than that in adjacent non-tumor mucosa (93.5%, 29/31) (P < 0.05). The expression of PTEN was negatively correlated with invasion and lymph node metastasis of gastric lymphoma(P < 0.05), while the Caspase-3 expression was negatively associated with the latter one(P < 0.05). Additionally, the PTEN expression was posi-tively correlated with Caspase-3 expression in the primary gastric malignant lymphoma(P < 0.05). Conclusions The down-regulated expression of PTEN and Caspase-3 played an important role in progression of primary malignant gastric lymphoma. PTEN, as a molecular marker of pathobiological behaviors of tumor, contributes to tumor progression by increasing cell mobility and angiogenesis, as well as decreasing cell adhesion and apoptosis.

Transcriptomic analysis reveals the effect of the exopolysaccharide of Psychrobacter sp.B-3 on gene expression in RAW264.7 macrophage cells

B-3 exopolysaccharide is extracted from the Antarctic psychrophilic bacterium Psychrobacter sp. B-3. We have previously shown that it activates macrophages and affects their immunoregulatory activities. To determine what genes are affected during this process, we detected the genes differentially expressed in cells of RAW264.7 macrophages treated with B-3 exopolysaccharide by transcriptomic analysis. B-3 exopolysaccharide treatment caused differential expression of 420 genes, of which 178 were up-regulated and 242 were down-regulated. These genes were shown to be involved in many aspects of cell function, mainly metabolism and immunity. Genes were enriched in multiple immune-related pathways, and the most significantly enriched genes were involved in antigen processing and presentation pathways. The pathway in which differentially expressed genes were the most significantly enriched was the metabolic pathway; specifically, the expression of many metabolic enzyme genes was altered by B-3 exopolysaccharide treatment. Additionally, the genes involved in metabolisms of amino acids, carbohydrates, lipids and nucleotides, varied to certain degrees. B-3 exopolysaccharide, therefore, appears to directly affect the immune function of RAW264.7 macrophages as an immunostimulant, or to indirectly change intracellular metabolism. This is the first study to determine the effect of an Antarctic psychrophilic bacterial exopolysaccharide on RAW264.7 macrophages. Our findings provide an important reference for research into the regulation of macrophage immune function by different polysaccharides.

Metabolic activity of Streptococcus mutans biofilms and gene expression during exposure to xylitol and sucrose

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media： Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy（CLSM）-based analyses of the microbial vitality, respiratory activity（5-cyano-2,3-ditolyl tetrazolium chloride, CTC） and production of extracellular polysaccharides（EPS） were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters： culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.

Study on differential gene expression profile of serum exosomes in patients with acute cerebral infarction

Objective To analyze the differential gene expression profile of serum exosomes in patients with acute cerebral infarction(ACI)and clarify the changes in gene expression related to cerebral infarction injury and the potential serum markers.Methods Four patients with ACI and five healthy people were enrolled in the PhaseⅠstudy.After serum isolation from peripheral blood,exosomes were extracted with exosomes kits,highthroughput detection of m RNA was performed with gene chips,and differentially expressed m RNAs were screened.Gene Ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed simultaneously.Furthermore,real-time polymerase chain reaction(q RT-PCR)was used to verify the expression levels of the screened differential m RNAs in the serum exosomes collected in PhaseⅡfrom 32 patients each in the ACI case and normal control groups.Results In the PhaseⅠstudy,there were 248 differentially expressed m RNAs(fold change≥2.0,P<0.05)among five patients in the normal control group and four patients in the case group,of which the expression of 242 was upregulated and that of six was downregulated.The results of GO functional enrichment analysis mainly included behavior regulation,cell connection,and antioxidant activity.The results of KEGG pathway enrichment analysis mainly included ribosomes,proteasomes,oxytocin signaling pathways,and oxidative phosphorylation.After researching and screening based on relevant literature,it was found that among the genes with significant differential expression,H3 F3 B m RNA may be associated with and might play an important role in ACI.The q RT-PCR method was used to detect the H3 F3 B mRNA expression in serum exosomes of 32 patients each in the normal control and case groups in PhaseⅡ;the expression was significantly higher in serum exosomes of the case group than in those of the normal control group(P<0.001).H3 F3 B mRNA expression in serum exosomes of the case group positively correlated with age,the National Institutes of Health Stroke Scale(NIHSS)score,and the maximum infarct size(P<0.05).Conclusion ACI can lead to changes in the serum exosomes mRNA expression profile,which may be closely related to the occurrence,development,and prognosis of this condition.These findings will provide direction for research on the molecular mechanism,diagnostic markers,and therapeutic targets of ACI.

Gene therapy with caspase-3 small interfering RNA-nanoparticles is neuroprotective after optic nerve damage

Apoptosis,a key mechanism of programmed cell death,is triggered by caspase-3 protein and lowering its levels with gene therapy may rescue cell death after central nervous system damage.We developed a novel,non-viral gene therapy to block caspase-3 gene expression using small interfering RNA(siRNA)delivered by polybutylcyanoacrylate nanoparticles(CaspNPs).In vitro CaspNPs significantly blocked caspase-3 protein expression in C6 cells,and when injected intraocularly in vivo,CaspNPs lowered retinal capsase-3 immunofluorescence by 57.9%in rats with optic nerve crush.Longitudinal,repeated retinal ganglion cell counts using confocal neuroimaging showed that post-traumatic cell loss after intraocular CaspNPs injection was only 36.1%versus 63.4%in lesioned controls.Because non-viral gene therapy with siRNA-nanoparticles can selectively silence caspace-3 gene expression and block apoptosis in post-mitotic neurons,siRNA delivery with nanoparticles may be promising for neuroprotection or restoration of central visual system damage and other neurological disorders.The animal study procedures were approved by the German National Act on the use of experimental animals(Ethic Committee Referat Verbraucherschutz,Veterinärangelegenheiten;Landesverwaltungsamt Sachsen-Anhalt,Halle,Germany,#IMP/G/01-1150/12 and#IMP/G/01-1469/17).

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective： To investigate the expression of Glypican-3 gene in （α-fetoprotein （AFP）-negative hepatocellular carcinoma （HCC） and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocelhllar carcinoma （HCC）, especially for AFP-negative ones. Methods： Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results： Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC （73.17%）. In contrast, Glypican-3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors （〉5 cm） （79.31%） and in small tumors （〈5 cm） （41.67%） （P〈0.01）. Gypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones （76.47% vs. 42.86%, P〈0.05）. The Glypican-3 expression was not correlated with age. sex. ttbsAg seropositivity, fibroeapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion： Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocareinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa

It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.

MP3RNA-seq:Massively parallel 3’end RNA sequencing for high-throughput gene expression profiling and genotyping

Transcriptome deep sequencing(RNA‐seq)hasbecome a routine method for global geneexpression profiling.However,its application tolarge‐scale experiments remains limited by costand labor constraints.Here we describe amassively parallel 3′end RNA‐seq(MP3RNA‐seq)method that introduces unique samplebarcodes during reverse transcription to permitsample pooling immediately following this initialstep.MP3RNA‐seq allows for handling of hun-dreds of samples in a single experiment,at acost of about$6 per sample for libraryconstruction and sequencing.MP3RNA‐seq iseffective for not only high‐throughput geneexpression profiling,but also genotyping.Todemonstrate its utility,we applied MP3RNA‐seqto 477 double haploid lines of maize.We iden-tified 19,429 genes expressed in at least 50%ofthe lines and 35,836 high‐quality singlenucleotide polymorphisms for genotypinganalysis.Armed with these data,we performedexpression and agronomic trait quantitativetrait locus(QTL)mapping and identified 25,797expression QTLs for 15,335 genes and 21 QTLsfor plant height,ear height,and relative earheight.We conclude that MP3RNA‐seq is highlyreproducible,accurate,and sensitive forhigh‐throughput gene expression profiling andgenotyping,and should be generally applicableto most eukaryotic species.

Genome-wide gene expression analysis for target genes to differentiate patients with intestinal tuberculosis and Crohn’s disease and discriminative value of FOXP3 mRNA expression

Background and aims:Crohn’s disease(CD)and intestinal tuberculosis(ITB)are both chronic granulomatous conditions with similar phenotypic presentations.Hence,there is need for a biomarker to differentiate between both these two diseases.This study aimed at genome-wide gene expression analysis of colonic biopsies from confirmed cases of ITB and CD in comparison with controls.To evaluate the role of T regulatory cells,forkhead box P3(FOXP3)mRNA expression was quantified in serum as well as in colonic biopsies from patients with ITB and with the controls.Methods:Paired samples,including serum and colonic biopsies,were taken from 33 study subjects(CD,ITB and controls),and total RNA was extracted.Human whole genome gene expression microarray analysis was performed using the Illumina HumanWG-6 BeadChip Kit with six total RNA samples of the three groups in duplicates.Real-time PCR for FOXP3 mRNA expression was analyzed in serum samples and colonic biopsy samples(4-CD,5-ITB,4-controls).Results:In CD and ITB there was 1.5-fold upregulation of 92 and 382 genes and 1.5-fold downregulation of 91 and 256 genes,respectively.Peroxisome proliferators via the PPARc pathway were most significantly downregulated(P<0.005)in CD.Additionally,the IL4/5/6 signaling pathways and Toll-like receptor signaling pathway were identified as significantly differentially regulated(P<0.005)at>2-fold change.In ITB,the complement activation pathway,specifically the classical pathway,was the most significantly upregulated.FOXP3 mRNA expression was significantly elevated in colonic biopsies obtained from ITB patients as compared with CD cases(4.7062.21 vs 1.4860.31,P=0.016).Conclusions:FOXP3 mRNA expression in colonic mucosa could be a discriminatory marker between ITB and CD.Upregulation of the complement activation pathway in ITB suggests that pathogenetic mechanisms for ITB are similar to those of pulmonary tuberculosis.In CD,downregulation of PPARc was seen in colonic tissue,suggesting that restoration of PPARc-dependent anti-microbial barrier function may be a therapeutic target.

Effect of antisense DNMT3b gene eukaryotic expression plasmid on expression of the DNMT3b gene in human biliary tract carcinoma cells

BACKGROUND: Hypermethylation of the promoter region is one of the major mechanisms of tumor suppressor gene inactivation. DNA methyltransferase 3b (DNMT3b), an enzyme that participates in the establishment of de novo methylation patterns, is highly expressed in many tumor cells and tissues, and it is closely associated with hypermethylation of the promoter of tumor suppressor genes. The aim of this study was to explore the effect of transfection with antisense DNMT3b gene eukaryotic expression plasmid on the expression of the DNMT3b gene in human biliary tract carcinoma cell. METHODS: The constructed antisense DNMT3b gene eukaryotic expression plasmid was transfected into the human biliary tract carcinoma cell line QBC-939 with lipofectamine transfection reagent, and positive cell clones were formed using G418 selection after transfection. The constructed recombinant plasmid was transfected into QBC-939 cells successfully and was confirmed by amplification of the exogenous neo^R gene with the polymerase chain reaction method. The expression of DNMT3b gene mRNA and protein was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry respectively. RESULTS: Following transfection, the mRNA level of the DNMT3b gene decreased from 0.956±0.053 to 0.209±0.023, and the protein level of the DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. Very significant differences were observed both at the transcription and posttranscription levels in the expression of the DNMT3b gene between the non-tranfection group and the antisense DN- MT3b gene eukaryotic expression plasmid transfection group (P<0.01). CONCLUSIONS: Transfection with the antisense DNMT3b gene eukaryotic expression plasmid can significantly reduce the expression level of the DNMT3b gene in the human biliary tract carcinoma cell line QBC-939. This study may provide a valid method to investigate the function of the DNMT3b gene and its role in biliary tract carcinoma.

Molecular Characterization of Cotton 14-3-3L Gene Preferentially Expressed During Fiber Elongation

The 14-3-3 protein, highly conserved in all eukaryotic cells, is an important regulatory protein. It plays an important role in the growth, amplification, apoptosis, signal transduction, and other crucial life activities of cells. A eDNA encoding a putative 14-3-3 protein was isolated from cotton fiber eDNA library. The eDNA, designated as Gh14-3-3L （Gossypium hirsutum 14-3-3-like）, is 1,029 bp in length （including a 762 bp long open reading frame and 5＇-/3＇-untranslated regions） and deduced a protein with 253 amino acids. The GhI4-3-3L shares higher homology with the known plant 14-3-3 proteins, and possesses the basic structure of 14-3-3 proteins： one dimeric domain, one phosphoralated-serine rich motif, four CC domains, and one EF Hand motif. Northern blotting analysis showed that Gh14-3-3L was predominantly expressed during early fiber development, and reached to the peak of expression in 10 days post anthers （DPA） fiber cells, suggesting that the gene may be involved in regulating fiber elongation. The gene is also expressed at higher level in both ovule and petal, but displays lower or undeteetable level of activity in other tissues of cotton.

Study of the SH3-domain GRB2-1ike 2 gene expression in laryngeal carcinoma

Background Laryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma （LSCC）. The abnormity of SH3-domain GRB2-1ike 2 （SH3GL2） gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC. Method Real-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC. Results The result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes. Conclusions These results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.