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Objective@#To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.@*Methods@#The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca2+ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.@*Results@#Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca2+ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05).@*Conclusion@#The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
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Objective To investigate the expression levels of Ezrin and AnnexinⅡ in gallbladder carci-noma and their association with clinicopathologic parameters and metastasis potential. Methods The tissue mi-croarray consisted of 59 gallbladder carcinoma tissues and 6 normal gallbladder tissues were examined for the ex-pression of Ezrin and AnnexinⅡusing immunohistochemistry technique. The expression of Ezrin and AnnexinⅡin 20 cases of fresh gallbladder carcinoma and 6 cases of normal gallbladder were measured with western blot. Results The expression of Ezrin and AnnexinⅡ were higher in the gallbladder cancer than those in the normal gallbladder tissue. The positive rate of Ezrin and AnnexinⅡ were 47.5% and 50.8% respectively. The expression of Ezrin was significantly correlated with live metastasis , lymph node metastasis and Nevin stages. The expression of AnnexinⅡwas significantly correlated with live metastasis , differentiation levels and Nevin stages. The expres-sion of Ezrin was correlated with AnnexinⅡ. Results of western blot suggested that Ezrin and Annexin II were highly expressed in gallbladder carcinoma tissues. The high expression of Ezrin and Annexin is closely related with liver invasion. Conclusion Measurement of the expression of Annexin and Ezrin II have important clinical significances to evaluate the malignant biological behavior of gallbladder carcinoma.
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Background and purpose:Traditional Chinese medicine with notable effect and little adverse reaction is increasingly concerned about the medical profession because of its great potential and advantage in treating pancreatic carcinoma. In this experiment, we studied the effects of oridonin on apoptosis and cytoskeletal protein F-actin in human pancreatic carcinoma SW1990 cells. Methods:SW1990 cells in culture medium were treated with different concentrations of oridonin. The inhibitory rate of the cells was measured by MTT assay. Morphology of cell apoptosis was observed by DAPI stain and cell apoptotic rate was detected by lfow cytometry (FCM). The morphological changes of F-actin were observed by laser confocal microscopy. Results:The growth of human pancreatic carcinoma SW1990 cells was signiifcantly inhibited by oridonin. Apoptosis morphological changes including condensation of chromatin and nuclear fragmentation were observed clearly by DAPI stain. The early apoptotic rate of SW1990 cells treated with 25, 50μmol/L oridonin was signiifcantly higher than that of the control group (3.78±0.46, 9.51±0.63 vs 0.73±0.06, P<0.05), and the late apoptotic rate and cell necrosis rate were also signiifcantly higher than that of the control group (14.40±1.78, 20.53±2.54 vs 4.16±0.31, P<0.05). F-actin was showed from polymerization to depolymerization after oridonin treatment. Conclusion:Oridonin can obviously inhibit the proliferation and induce apoptosis of SW1990 cells. The mechanisms may involve the depolymerization of F-actin after treatment with oridonin.
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Objective To investigate the effects of chronic ethanol exposure and withdrawal on the expression of actin-binding protein cofilin,p-cofilin and cyclin-dependent kinase-5 (cdk5) in the nucleus accumbens and striatum in rat brain.Methods Twenty-four male SD rats were randomly divided into one control group and three experimental groups.In the experimental groups,ethanol was administered in drinking water at the concentration of 6％ (V/V) for two months.Rats in control group drank normal drinking water.After two months ethanol was removed and ethanol withdrawal syndromes were evaluated.Rats were sacrificed on withdrawal 0 h,withdrawal 6 h and withdrawal 2 d.The expression levels of cofilin,p-cofilin(ser3)and cdk5 in the rat brain were measured by immunohistochemistry methods.Results Withdrawal syndrome scores of ethanol fed rats were obviously higher than those of control rats after ethanol was removed,the highest score occurred at 6 h after ethanol withdrawal.In the nucleus accumbens area of rat brain,the levels of cofilin on withdrawal 0 h significantly decreased compared with control group ((0.31±0.05),(0.39± 0.05),P＜ 0.05).The levels of cdk5 on withdrawal 0 h and withdrawal 6 h significantly increased compared with control group((0.36±0.07),(0.34±0.07),(0.25±0.05),P＜0.05).In the striatum of rat brain,the levels of cofilin on withdrawal 0 h significantly decreased compared with control group ((0.26±0.04),(0.34±0.05),P＜0.05).The levels of p-cofilin on withdrawal 6 h significantly increased compared with control group((0.43±0.06),(0.30±0.06),P＜0.01).The levels of cdk5 on withdrawal 0 h significantly increased compared with control group((0.35±0.06),(0.26±0.05),P＜0.05),and the levels of cofilin on withdrawal 6 h significantly decreased compared with control group((0.37±0.06),(0.26±0.05),P＜0.01).Conclusion Chronic ethanol exposure can induce the development of ethanol dependence,and it accompanies with changes in the expression of actin-binding protein and cdk5 in the brain of rats.
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Background Primary open angle glaucoma (POAG) is one of the frequent glaucomatous types,and genetic factor participates in pathogenesis and development of the disease.Recently,MYOC mutation was found to be associated with POAG.Objective This study was to describe the clinical and genetic findings in a POAG family from Luoyang,China.Methods This study protocol was approved by Ethic Committee of Affiliated First Hospital of Henan University of Science and Technology.The study adhered to Declaration of Helsinki.A POAG family with 29 members of 5 generations was surveyed and followed-up for 5-year duration.The mode of inheritance was determined by the pedigree analysis.The periphery blood sample was collected form 12 families and 100 health controls for the extraction of genomic DNA under the informed consent.The third exon and its flanking introns of MYOC were amplified,and quantitative real time PCR products were sequenced,and the structure and function of mutated gene were examined by restriction fragment length polymorphism analysis.The predicted effects of the detected variants on the secondary structure of MYOC protein were evaluated using Garnier-Osguthorpe-Robson (GOR) method,and homology analysis of protein was carried out by Blast software provided by National Center for Biotechnology Information (NCBI).Results This POAG family included 29 members of 5 generations,and the clinical data were not clear in 11 family members.Three individuals from 3 generations were determined POAG,another one was ocular hypertension,and 2 were carriers.Pedigree analysis appeared an autosomal dominant inheritance.In 12 subjects included 6 members genetically affected and 6 members with normal phenotype,the heterozygous mutation was found in the third exon of MYOC gene in 6 genetically affected members,which revealed a T→C transition at position 1021 (p.S341P),resulting in a switch of serine (Ser) to proline (Pro).It was a missense mutation abolished a CviKI-1 restriction site that segregated with the affected members.Secondary structure prediction of p.S341P suggested that myocilin protein was misfolded.Analysis of protein homology and switched Ser was conservative amine acid at position 1021 (p.S341P).No similar change was found in the 6 normal families and the normal controls.Conclusions Ser341Pro MYOC mutation is disease-causing factor in the POAG family of Luoyang.The clinical and genetic features of this mutation warrant further investigation.The mutation spectrum of MYOC is expanded to offer a better diagnosis and treatment for POAG patients.
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AIM: To study changes of expression of muscle LIM protein(MLP) mRNA and protein during the progression of left ventricular hypertrophy induced by coarctation of abdominal aorta in rats and to explore if there is disruption of cytoskeleton protein during hypertrophic growth in left ventricle. METHODS: Using a model of hypertrophy induced by coarctation of abdominal aorta(CAA) in male Wistar rats, hemodynamics, ventricular hypertrophic index, expression of MLP mRNA and protein were investigated in the experimental animals at 1, 4, 8, 16 weeks after operation and sham operation. RESULTS: Left ventricular shows significantly hypertrophy 4 weeks after operation ( P0.05 ). CONCLUSIONS: MLP changed at transcription level and there was no disruption of cytoskeleton during hypertrophic growth of left ventricle without sever heart failure. As the actin-base cytoskeleton protein, MLP plays a critical role in the maintenance of cardiac myocyte cytoskeleton and cardiac systolic function.
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BACKGROUND: Cerebral cortical dysplasia (CD) is one of the important causes of intractable epilepsies and characterized histologically by disorganized cortical lamination and cytomegalic dysplastic neurons. Although various cytoskeletal abnormalities have been found in dysplastic neurons of CD, the pathogenetic role of dysplastic neurons has rarely been investigated. METHODS: In this study, immunohistochemical analysis was performed using antibodies against non-phosphorylated high- or medium-molecular weight neurofilament protein and microtubule-associated protein 2 (MAP-2) in surgical specimens of CD. In order to know the possible relationship of dysplastic neurons with cytoskeletal abnormalities and various neurotrophin receptors, NGFR p75, trkA, trkB, and trkC immunoreactivities were also analyzed. RESULTS: Dysplastic neurons showed strong immunoreactivities for non-phosphorylated high- or medium-molecular weight neurofilament protein and MAP-2, which might reflect abnormal outgrowth and altered plasticity of the dysplastic neurons. TrkB and trkC were strongly expressed in dysplastic neurons and NGFR p75 was also strongly expressed in some dysplastic neurons. CONCLUSIONS: Since it has been known that brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have effects on the differentiation of neuronal precursor cells from the cortex and on dendritic and axonal arborization, increased expression of trkB and trkC may play a role in cytoskeletal abnormalities and altered synaptic transmission in dysplastic neurons.