Effect of ACTN4 on cell proliferation of esophageal squamous cell carcinoma by targeting NDUFV1 / 安徽医科大学学报

Objective @#To investigate the expression of alpha-actinin-4 (ACTN4) and the effect on cell proliferation in esophageal squamous cell carcinoma (ESCC) ．@*Methods @#The expression of ACTN4 in ESCC tissues and paired normal tissues was detected by immunohistochemistry，and the correlation between ACTN4 and the clinicopathologi- cal features was analyzed statistically．The ACTN 4 shRNA lentiviral plasmids were constructed ，and the stable ECA109 strain with ACTN4 shRNA knockdown was established using lentivirus packaging technology．The knock- down efficiency on protein level was checked by Western blot，and cell proliferation was detected by colony forma- tion assays．The downstream target proteins were validated in ESCC cell line ECA109 based on the previous pro- teomics analyses in melanoma cell line A375 with or without ACTN4 shRNA knockdown. @*Results @#The expression of ACTN4 in ESCC tissues was significantly higher than that of normal tissues．ACTN4 shRNA stable knockdown ECA109 cell strains were successfully constructed． The results of colony formation assays showed that ACTN4 knockdown inhibited the cell proliferation and down-regulated NADH ∶ Ubiquinone oxidoreductase core subunit V1 ( NDUFV1) protein expression in ECA109 cells．@*Conclusion @#Upregulation of ACTN4 in ESCC cells promotes the cell proliferation and enhances the protein expression of NDUFV1 .

Mutations of ACTN4 and SYNPO genes promoter in primary focal segmental glomerulosclerosis / 中华肾脏病杂志

Objective To investigate the mutations ACTN4 and SYNPO genes promoter in sporadic primary focal segmental glomerulosclerosis (FSGS) and to analyze the role of mutations in FSGS. Methods The study consisted of 82 Chinese primary FSGS, including 39 females and 43 males, ranged from 12 to 76 years old. Seventy volunteers were selected as healthy control group. Genomie DNA was extracted from peripheral blood cells of FSGS patients and hair of patients' parents by polymerase chain reaction (PCR) and direct sequencing to analyze ACTN4 and SYNPO gene promoter mutations. Mutations were matched with GenBank and TRANSFAC software database (www.ncbi.nlm.nih.gov; www.genometix.de; www.gene-regulation, corn). Dual luciferase assay system was used to analyze the promoter region mutations, based on PGL3-Basie vector, pRL-SV40 and PCI2 cell line. Hair DNA of novel mutation patients' parents was sequenced. Expression of alpha-actinin-4 and synaptopodin in patients' kidney tissue was examined by immunofluorescence. Results Three patients with 1-34C>T, 1-590delA and (1-1044delT)+ (I-797T >C) +(1-769A >G) heterozygous mutations were found in ACTN4 gene promoter respectively, and two patients with 1-24G>A and 1-851C>T heterozygous mutations in SYNPO gene promoter respectively. The same mutations were not found in the control group of 70 healthy people. Except one patient accepting her parents' 1-1044delT and 1-797T>C mutated chromosome respectively, no same mutations were found in patients' parents. Protein expression of alpha-actinin-4 and synaptopodin was reduced in mutated patients' kidneys. Except 1-1044delT group, luciferase activity in mutated groups decreased. (1-1044delT)+(1-797T>C)+(1-769A>G) mutation was associated with poor outcome and patient with these mutations progressed to end-stage renal failure. Conclusion Mutations of ACTN4 and SYNPO gene promoters affect gene transcription and protein translation, which may contribute to the onset of sporadic primary FSGS.

Angiotensin II Effect on alpha-Actinin in Glomerular Epithelial Cells / 대한신장학회잡지

BACKGROUND: Angiotensin II plays a potential role in renal injury not only by its vasoconstrictive effects but also its biochemical effects. alpha-Actinin, an actin-linked glycoprotein, is expressed in podocytes and known to be rearranged and changed in various glomerular diseases. We investigated the effect of angiotensin II on the alpha-actinin in the glomerular epithelial cells to find out the fact that it could be prevented by losartan, a type 1 angiotensin receptor antagonist. METHODS: Glomerular epithelial cells were treated with various concentrations of angiotensin II in culture media, and then we compared the localization and amount of alpha-actinin by confocal microscopy and Western blot analysis, respectively. We also compared the differences in the localization and protein amount of alpha-actinin by various concentrations of losartan in the presence of angiotensin II. In addition, we tried to observe the mRNA expression of alpha-actinin via RT-PCR. RESULTS: The fluorescent and band intensities of alpha-actinin were decreased by angiotensin II in a dose-dependent manner by confocal microscopy and Western blot analysis, respectively. These changes of alpha-actinin by angiotensin II were reversed by losartan in dose dependent manner. Angiotensin II also changed the distribution of alpha-actinin from peripheral to inner cytoplasm in dose-dependent manner, which was also reversed by losartan. The different expression of alpha-actinin m-RNA by RT-PCR were unremarkable. CONCLUSIONS: Angiotensin II decreases the amount of alpha-actinin protein and and makes cytoskeletal changes in glomerular epithelial cells, which could be reversed by losartan. It suggests that it could be prevented by angiotensin II AT1 receptor blockers.

Angiotensin II Effect on alpha-Actinin in Glomerular Epithelial Cells / 대한신장학회잡지

BACKGROUND: Angiotensin II plays a potential role in renal injury not only by its vasoconstrictive effects but also its biochemical effects. alpha-Actinin, an actin-linked glycoprotein, is expressed in podocytes and known to be rearranged and changed in various glomerular diseases. We investigated the effect of angiotensin II on the alpha-actinin in the glomerular epithelial cells to find out the fact that it could be prevented by losartan, a type 1 angiotensin receptor antagonist. METHODS: Glomerular epithelial cells were treated with various concentrations of angiotensin II in culture media, and then we compared the localization and amount of alpha-actinin by confocal microscopy and Western blot analysis, respectively. We also compared the differences in the localization and protein amount of alpha-actinin by various concentrations of losartan in the presence of angiotensin II. In addition, we tried to observe the mRNA expression of alpha-actinin via RT-PCR. RESULTS: The fluorescent and band intensities of alpha-actinin were decreased by angiotensin II in a dose-dependent manner by confocal microscopy and Western blot analysis, respectively. These changes of alpha-actinin by angiotensin II were reversed by losartan in dose dependent manner. Angiotensin II also changed the distribution of alpha-actinin from peripheral to inner cytoplasm in dose-dependent manner, which was also reversed by losartan. The different expression of alpha-actinin m-RNA by RT-PCR were unremarkable. CONCLUSIONS: Angiotensin II decreases the amount of alpha-actinin protein and and makes cytoskeletal changes in glomerular epithelial cells, which could be reversed by losartan. It suggests that it could be prevented by angiotensin II AT1 receptor blockers.

Cytoplasmic Domain of Intercellular Adhesion Molecule-1(ICAM-1)is Important for the ICAM-1 Mediated Membrane Projection of Endothelial Cells / 대한해부학회지

Intercellular adhesion molecule-1 (ICAM-1)has been shown to enhance leukocyte adhesion, thereby inducing migration through blood endothelial cells. However, the molecular event during the process of adhesion is largely unknown. To examine the role of ICAM-1 cytoplasmic domain in SDF-1 alpha-induced T lymphocyte migration and adhesion, mutant human ICAM-1 molecules were expressed in COS-7 cell line. COS-7 cells expressing ICAM-1_GFP mutant without alpha-actinin revealed no association with the actin cytoskeleton, while wild-type ICAM-showed clear association with the actin, as observed by confocal microscopy, suggesting that actinin binding motif in the cytoplasmic domain of ICAM-1 is important for the proper localization of ICAM-1 on the cell membrane. However, based on adhesion assay, we found that the cytoplasmic domain of ICAM-1 is not essential for the binding of lymphocytes which were activated by SDF-1alpha. On the other hand, ICAM-1-mediated receptor-ligand clustering event was significantly inhibited in the cells expressing ICAM-1 mutants without alpha-actinin or whole cytoplasmic domain. Taken together, these results suggest that ICAM-1 cytoplasmic domain is not essential for the adhesion but important for the ligand-receptor-mediated membrane projection of endothelial cells before trans-endothelial migration of lymphocytes.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on the alpha-actinin-4 Expressed by Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in GEpC with retraction and effacement of the highly specialized interdigitating podocyte foot processes. I examined the molecular basis for this alteration of the podocyte phenotype, involving cytoskeletal changes especially on alpha-actinin-4 as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration and regulation of the podocyte actin cytoskeleton. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (30 mM) and AGE- or BSA-added conditions and examined the distribution of alpha-actinin-4 by confocal microscope and measured the change of alpha-actinin-4 production by Western blotting and RT-PCR. RESULTS: I found that alpha-actinin-4 moved from peripheral cytoplasm to inner actin filaments complexes in the condition of AGE and high glucose by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the alpha-actinin-4 productions by 22.3% (p>0.05) and 28.1% (p<0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of alpha-actinin-4 more significantly by 53.6% compared to those of control (p<0.01). S uch changes could not be seen by osmotic control. The expression of mRNA for alpha- actinin-4 were not changed in condition of high glucose or AGE-coated surface, however, both high glucose and AGE significantly decreased the expression of alpha-actinin-4 mRNA by 15.7% compared to those of control. CONCLUSION: I could suggest that both high glucose and AGE induce the cytoplasmic translocation and suppress the production of alpha-actinin-4 at transcriptional level and these changes may explain the cytoskeletal changes of GEpC in diabetic conditions.

A Study on the Proteins that Interact with Human Nebulin SH3 Domain / 대한정형외과연구학회지

OBJECTIVE: bjective: By identifying the unknown substance responsible for binding with nebulin SH3 domain within the sarcomeric Z-line, we tried to find out Z-line structure which plays an important role on muscle contraction and maintenance of muscle funtion. METHOD: First, the bait plasmid was made by binding the DNA binding domain of Gal4 protein of yeast and the SH3 domain. Second, library plasmid was made by binding activation domain and human skeletal cDNA library. Then, the base sequence of the clone, produced by combining the two proteins expressed by transgenically converted plasmid in yeast, was analyzed. RESULT: We screened out six true positive clones and analyzed the base sequence of the two of six clones. We identified them to be alpha-actinin2. CONCLUSION: We can theorize that Neublin SH3 domain and alpha-actinin2 plays a vital role for the integration of Z-line. Thus, this is an important data in further studying muscle functions, mechanisms, and muscular disease as well.

Morphological Observations on the Changes of Membrane Permeability by Immobilization Stress in the Rat Atrial Myocyte / 대한해부학회지

This study was performed to investigate the effect of immobilization stress on the ultrastructural changes and membrane permeability in rat atrial myocyte using immunohistochemical and lanthanum tracer techniques. Male Sprague-Dawley rats, body weight 160~200 g, were used for all immobilization stress group. Rats were immobilized in small round plastic tube for 6, 12 or 24 hours, except for the control group. Alterations of myocardial myoglobin and alpha-actinin as well as membrane permeability after immobilization stress were examined by immunohistochemistry, and lanthanum permeability of the rat atrial myocyte were observed by electron microscopy. In the control group, there was no loss of myoglobin or alpha-actinin from the atrial myocytes. After 6 and 12 hours immobilization stress, the loss of myoglobin and alpha-actinin could be identified the atrial myocytes. In the 24 hour immobilization groups, the content of the myoglobin and alpha-actinin recovered partially. Lanthanum was deposited only in the intercellular space of the atrial myocardium in the control group. In the 6 hour immobilization group, the atrial myocytes showed severe ultrastructural changes during immobilization stress. Lanthanum deposited in the sarcoplasm, myofibrils, adjacent of mitochondria, and mitochondrial matrix. In the 12 or 24 hour immobilization groups, the morphological alteration of atrial myocytes appeared weekly. In the 12 hour group, lanthanum deposited in myofibrils, adjacent of mitochondria and in the mitochondrial matrix. In the 24 hour group, lanthanum deposited mainly in intercellular space of atrial myocardium, and rarely in the sarcoplasm of myocytes. These results suggest that the immobilization stress may induce the alteration of cardiac cell membrane permeability and the ultrastructures of atrial myocardium.

Localization of cytoskeletal proteins in Cryptosporidium parvum using double immunogold labeling

Actin and some actin binding proteins such as tropomyosin, -actinin and troponin T were localized by simultaneous double immunogold labeling in several developmental stages of Cryptosporidium parvum. All of the observed developmental stages have many particles of tropomyosin and actin around pellicle and cytoplasm. Tropomyosin was labeled much more than the actin when these two proteins were labeled simultaneously. And alpha-actinin was labeled mostly in the pellicle, but troponin T labeling was very rarely observed. From this study, it was suggested that tropomyosin seems to be one of the major proteins of C. parvum, so it must be playing important roles in C. parvum.