R577x of the actn3 gene as predictor of physical performance in ultramarathon runners / R577x do gene actn3 como preditor de desempenho físico em ultramaratonistas / R577x del gen actn3 como predictor de desempeño físico en ultramaratonistas

ABSTRACT Introduction: Genetic factors appear to explain why some athletes perform better in competition and training than their peers. Objective: To determine the occurrence of R577X polymorphism of the ACTN3 gene in mountain runners. Methods: The sample consisted of 19 female mountain runners with a mean age of 41.2 ± 6.1 years. Genotyping of R577X polymorphism of the ACTN3 gene was performed by the polymerase chain reaction (PCR) method with DNA extracted from saliva. The genotypic and allelic frequencies of the athletes were evaluated and compared with data from the literature. Hardy-Weinberg equilibrium and Chi-square with Yates correction were used, with a significance level of p<0.05. Results: The genotypic distributions did not show any significant differences between the athletes and the control group, with RR = 15.8%, RX = 57.9%, XX = 26.3%. In regard to allelic distribution, the nonfunctional allele was higher in the study group than in the control group, with R = 44.7%, X = 55.3% for p = 0.0350. Conclusion: The data revealed a possible relationship between the ACTN3 X allele and athletic performance in Brazilian female mountain runners. Level of evidence II; Development of diagnostic criteria in consecutive patients (with "gold" reference standard applied).

RESUMO Introdução: O componente genético parece explicar a melhor adaptação à competição e ao treinamento de alguns atletas com relação a seus pares. Objetivo: Determinar a ocorrência do polimorfismo R577X do gene ACTN3 em atletas corredoras de montanha. Métodos: A amostra foi composta por 19 corredoras de montanha com média de idade de 41,2 ± 6,1 anos. A genotipagem do polimorfismo R577X do gene ACTN3 foi realizada pelo método de reação em cadeia de polimerase (PCR) com DNA extraído da saliva. As frequências genotípica e alélica das atletas foram avaliadas e comparadas com dados da literatura: Equilíbrio de Hardy-Weinberg, Qui-quadrado com correção de Yates, sendo adotado como nível de significância p<0,05. Resultados: As distribuições genotípicas RR = 15,8%, RX = 57,9%, XX = 26,3% em comparação com o grupo controle e as atletas da presente investigação, não mostraram diferença significativa. Quanto à distribuição alélica, o alelo não funcional no grupo estudado com relação ao controle foi maior R = 44,7%, X = 55,3% para p = 0,0350. Conclusão: Os dados revelaram uma possível relação entre o alelo X do ACTN3 e a condição atlética em corredoras de montanha brasileiras. Nível de evidência II; Desenvolvimento de critérios diagnósticos em pacientes consecutivos (com padrão de referência "ouro" aplicado).

RESUMEN Introducción: El componente genético parece explicar la mejor adaptación a la competición y al entrenamiento de algunos atletas con relación a sus pares. Objetivo: Determinar la ocurrencia del polimorfismo R577X del gen ACTN3 en atletas corredoras de montaña. Métodos: La muestra fue compuesta por 19 corredoras de montaña con promedio de edad de 41,2 ± 6,1 años. El genotipado del polimorfismo R577X del gen ACTN3 fue realizado a través del método de reacción en cadena de polimerasa (PCR), con ADN extraído de la saliva. Las frecuencias genotípica y alélica de las atletas fueron evaluadas y comparadas con datos de la literatura: Equilibrio de Hardy-Weinberg, Chi-cuadrado con corrección de Yates, siendo adoptado como nivel de significancia p<0,05. Resultados: Las distribuciones genotípicas RR = 15,8%, RX = 57,9%, XX = 26,3% en comparación con el grupo control y las atletas de la presente investigación, no mostraron diferencia significativa. En referencia a la distribución alélica, el alelo no funcional en el grupo estudiado con relación al control fue mayor R = 44,7%, X = 55,3% para p = 0,0350. Conclusión: Los datos revelaron una posible relación entre el alelo X del ACTN3 y la condición atlética en corredoras de montaña brasileñas. Nivel de Evidencia II; Desarrollo de criterios diagnósticos en pacientes consecutivos (con estándar de referencia "oro" aplicado).

Polimorfismo genético ACTN3 R577X en deportistas universitarios chilenos / ACTN3 R577X gene polymorphism in Chilean college athletes

Uno de los principales factores genéticos que influenciarían el rendimiento muscular humano es el gen ACTN3, que codifica la proteína estructural del sarcómero α-actinina-3. El polimorfismo R577X (rs1815739) del gen ACTN3 ha sido asociado con varios indicadores de rendimiento muscular y físico en deportistas y población general, pero este fenómeno ha sido escasamente descrito en poblaciones de Latinoamérica y Chile. Por lo tanto, el objetivo del presente estudio fue describir la frecuencia genotípica y distribución alélica de los genotipos de ACTN3 R577X en deportistas universitarios chilenos. 129 deportistas universitarios chilenos representantes de diferentes selecciones deportivas (halterofilia, balonmano, voleibol, rugby, basquetbol, futbol y futsal) participaron como voluntarios. Los análisis moleculares del polimorfismo R577X del gen ACTN3 fueron realizados mediante reacción en cadena de la polimerasa (PCR) y restricción enzimática (RFLP). La distribución de genotipos del polimorfismo ACTN3 R577X fue RR: 34,8 % (n=45), RX: 50,4 % (n=65), XX: 14,7 % (n=19), y la frecuencia relativa de alelos fue R: 0,601 y X: 0,399. Además, se encontró asociación entre distribución de genotipos (c2= 12,26; 2 gl; p=0,002) y frecuencia relativa de alelos (c2= 11.02; 1 gl; p=0.0009) con el sexo de los participantes. Sin embargo, no hubo asociación al realizar análisis por tipo de deporte practicado. Los hallazgos de la presente investigación sugieren que el polimorfismo R577X del gen ACTN3 está asociado con el sexo en deportistas universitarios chilenos. Además, estos resultados describen de forma inédita la distribución genotípica y frecuencia alélica de esta variante genética en población chilena, mostrando una distribución similar a otros estudios realizados en poblaciones de deportistas en Brasil, Rusia, Estados Unidos y Turquía. No obstante, también muestra diferencias con otras poblaciones generales y de deportistas.

One of the main genetic factors that influence the muscular performance is the gene that encodes the structural protein α-actinin-3 (ACTN3). The R577X polymorphism (rs1815739) of ACTN3 has been associated with indicators of muscle and physical performance in athletes and general population, but this has been scarcely described in the Latin American and Chilean population. Thus, the aim of the present study was to describe the genotypic frequency and allelic distribution of ACTN3 R577X genotypes in college athletes. A total of 129 unrelated Chilean college athletes representing various sport disciplines (weightlifting, handball, volleyball, rugby, basketball, soccer and futsal) were volunteered for the study. ACTN3 R577X gene polymorphism was analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). For the total sample the genotypes distribution for R577X polymorphism was RR: 34.8 % (n=45), RX: 50.4 % (n=65), XX: 14.7 % (n=19), and the relative frequency of alleles was R: 0,601 and X: 0,399. Moreover, an association was found between genotype distribution (c2= 12.26; 2 df; p=0.002) and allele frequencies (c2= 11.02; 1 df; p=0.0009) with the sex of the participants. However, there were no associations when performing analysis by type of sports. These findings suggest that the R577X polymorphism of the ACTN3 gene is associated with sex in Chilean college athletes. Furthermore, these results describe in an unprecedented manner, the genotypic distribution and allelic frequency of this genetic variant in Chilean population, showing a similar distribution to other studies conducted in populations of athletes in Brazil, Russia, the United States and Turkey. However, it also shows differences with other general and athletes populations.

Mutational analysis of 3 Glanzmann's thrombasthenia pedigrees / 中华检验医学杂志

Objective To explore the molecular pathogenesis of 3 Glanzmann's thrombasthenia pedigree by using bioinformatics software and provide evidence for in vitro experiments. Methods The genetic analysis of 3 pedigree diagnosed as Glanzmann's thrombasthenia was carried out. Clustalx-2.1 win software was used to analyze the conservatism of mutant sites in homologous sequences. Bioinformatics software such as PolyPhen-2, PROVEAN, SIFT and Mutationtaster was used to analyze the biological effect of mutation. SPDBV software constructed the molecular structure model of mutant protein and evaluated the influence of mutation on protein structure. Results The "new mutations" found in 3 Glanzmann's thrombasthenia pedigree were ITGA2B:c. 814G>C (p. Val272Leu), ITGA2B:c. 432G>A (p. Trp144Ter) and ACTN1:c. 2458A>G (p. Ile820Val). All three mutations were highly conserved among homologous species. Mutationtaster software showed that 3 new mutations were likely pathogenic. PolyPhen-2 and PROVEAN software showed ITGA2B p.Val272Leu and ACTN1 p.Ile820Val were benign and SIFT software showed that ITGA2B p. Val272Leu were likely pathogenic, while ACTN1 p. Ile820Val is benign. The result of SPDBV software showed that the Val272 of ITGA2B was transformed to Leu, neutralizing all the original hydrogen bond. The Trp144 of ITGA2B is transformed to Ter, resulting in the truncated proteins with only 113 amino acid residues. All these mutations affected the molecular structure of GPⅡb, resulting in a decrease ofGPⅡb/Ⅲa expression. When the Ile820 of ACTN1 is transformed to Val, onlyretained the hydrogen bond of Ile820 and Asp822, neutralized the rest hydrogen bond, whichaffected the molecular structure and protein function of ACTN1. Conclusion The mutations of ITGA2B:c.814G>C (p.VAL272LEU), ITGA2B:c.432G>A (p.Trp144Ter) and ACTN1:c.2458A>G (p.Ile820Val) are pathogenic.

Effect of Tacrolimus on the expression of α-actinin-4 in podocytes induced by Puromycin aminonucleoside / 中华实用儿科临床杂志

Objective@#To investigate the effects of Tacrolimus(FK506) and Puromycin aminonucleoside(PAN) on apoptosis and expression of α-actinin-4 mRNA and protein in mouse glomerular podocytes in order to explore the protective effect of FK506 on podocytes.@*Methods@#Mouse glomerular podocytes were cultured in vitro, and the control group, PAN group and FK506 group were established.After 8 h, 24 h and 48 h of treatment, the cell morphology was observed and the apoptosis rate was detected.The cells were collected by real-time PCR and Western blot was used to detect the mRNA and protein expression of α-actinin-4.@*Results@#The cell body area of the PAN group was significantly smaller than that of the control group, and the cell area of the FK506 group was larger than that of the PAN group.There was no significant difference in the rate of podocyte apoptosis between those groups at 8 h (all P>0.05). At 24 h and 48 h, the apoptotic rate of podocytes in PAN group[(8.21±0.41)%, (16.32±0.17)%] were significantly higher than those in the control group[(4.28±0.35)%, (6.27±0.28)%], and the differences were significant (all P<0.05). The apoptosis rate of podocytes in FK506 group[(6.26±0.24)%, (13.32±0.24)%] were significantly lower than those in PAN group, and the differences were significant (all P<0.05). At 8 h, there was no significant difference in the expression of α-actinin-4 mRNA and protein(all P>0.05). The expression of mRNA (2.42±0.21, 3.78±0.25) and protein(0.77±0.04, 1.22±0.10) in the PAN group was significantly higher than mRNA(1.50±0.22, 2.15±0.15) and protein(0.44±0.03, 0.83±0.07) in the control group at 24 h and 48 h, and the differences were significant (all P<0.01). The expression of mRNA (1.65±0.24, 1.70±0.32) and protein (0.52±0.05, 0.56±0.07) in FK506 group was significantly lower than that of PAN group, and the differences were significant (all P<0.05).@*Conclusions@#FK506 can effectively inhibit the damage of PAN on podocytes and stabilize the expression of α-actinin-4, which provides a basis for the clinical application of FK506 in the treatment of glomerular diseases.

Effect of Tacrolimus on the eXpression of α-actinin-4 in podocytes induced by Puromycin aminonucleoside / 中华实用儿科临床杂志

Objective To investigate the effects of Tacrolimus( FK506 )and Puromycin aminonucleoside ( PAN)on apoptosis and expression of α-actinin-4 mRNA and protein in mouse glomerular podocytes in order to ex-plore the protective effect of FK506 on podocytes. Methods Mouse glomerular podocytes were cultured in υitro,and the control group,PAN group and FK506 group were established. After 8 h,24 h and 48 h of treatment,the cell mor-phology was observed and the apoptosis rate was detected. The cells were collected by real-time PCR and Western blot was used to detect the mRNA and protein expression of α-actinin-4. Results The cell body area of the PAN group was significantly smaller than that of the control group,and the cell area of the FK506 group was larger than that of the PAN group. There was no significant difference in the rate of podocyte apoptosis between those groups at 8 h( all P>0. 05). At 24 h and 48 h,the apoptotic rate of podocytes in PAN group[(8. 21 ± 0. 41)%,(16. 32 ± 0. 17)%]were significantly higher than those in the control group[(4. 28 ± 0. 35)%,(6. 27 ± 0. 28)%],and the differences were significant(all P<0. 05). The apoptosis rate of podocytes in FK506 group[(6. 26 ± 0. 24)%,(13. 32 ± 0. 24)%] were significantly lower than those in PAN group,and the differences were significant(all P<0. 05). At 8 h,there was no significant difference in the expression of α -actinin -4 mRNA and protein( all P >0. 05 ). The expression of mRNA(2. 42 ± 0. 21,3. 78 ± 0. 25)and protein(0. 77 ± 0. 04,1. 22 ± 0. 10)in the PAN group was significantly higher than mRNA(1. 50 ± 0. 22,2. 15 ± 0. 15)and protein(0. 44 ± 0. 03,0. 83 ± 0. 07)in the control group at 24 h and 48 h,and the differences were significant(all P<0. 01). The expression of mRNA(1. 65 ± 0. 24,1. 70 ± 0. 32)and protein(0. 52 ± 0. 05,0. 56 ± 0. 07)in FK506 group was significantly lower than that of PAN group,and the differ-ences were significant(all P<0. 05). Conclusions FK506 can effectively inhibit the damage of PAN on podocytes and stabilize the expression of α-actinin-4,which provides a basis for the clinical application of FK506 in the treat-ment of glomerular diseases.

Association of actn3 r577x and ace i/d polymorphisms in brazilians wrestlers / Associação dos polimorfismos actn3 r577x e ace i/d em lutadores brasileiros / Asociación de los polimorfismos actn3 r577x y ace i/d en luchadores brasileños

ABSTRACT Introduction: By associating genetics and sport, it is possible to identify subjects with greater capacity to adapt to training, and lower chances of injury. Objective: The investigation evaluated the genotypic and allelic distribution of ACTN3 R577X and ACE I/D polymorphisms in Brazilian high-performance athletes in wrestling and percussion combat sports. Methods: The study included 37 male athletes ranked from first to third place in world scenarios, divided into two groups: wrestling (23 wrestlers, being 11 of Judo, 4 of Greco-Roman style, 8 of Brazilian Jiu Jitsu, with mean age of 27.3 ± 6.9 years) and percussion combat sports (14 athletes with a mean age of 25.7±4.4 years, being 6 of Karate, 3 of Muay Thai, 4 of Taekwondo, 1 Boxing). Genotyping of ACTN3 and ACE I/D polymorphisms was performed by polymerase chain reaction (PCR) from the genomic DNA. Genotypic and allelic distributions were compared with control populations and athletes by Chi-square test and Fisher's exact test; all analyzes considered p ≤ 0.05. Results: The genotypic distributions and allelic frequencies of ACTN3 RR=46%, RX=38% and XX=16%; R=65% and X=35%, and ACE I/D DD=47.7%, ID=34.3% and II=20%; D=62.9% and I=37.1% did not differ from the control population; however, when compared with wrestling athletes a significant difference was observed. Conclusion: These results suggest an association of ACTN3 R577X and ACE I/D genes with Brazilian high-performance wrestling athletes.

RESUMO Introdução: Ao associar genética e esporte, existe a possibilidade de identificar sujeitos com maior capacidade de adaptação ao treinamento com menores chances de lesões. Objetivo: A investigação avaliou a distribuição genotípica e alélica dos polimorfismos ACTN3 R577X e ACE I/D em lutadores de domínio e percussão de alto rendimento brasileiros. Métodos: Participaram do estudo 37 atletas do sexo masculino, colocados de primeiro a terceiro lugar nos cenários mundiais, divididos em dois grupos: domínio (23 lutadores sendo 11 de judô, 4 de luta greco-romana, 8 de jiu-jitsu brasileiro; a média de idade foi 27,3 ± 6,9 anos) e percussão (14 atletas com média de idade de 25,7 ± 4,4 anos sendo 6 de caratê, 3 de muay thai, 4 de taekwondo, 1 de boxe). A genotipagem dos polimorfismos do ACTN3 e ACE I/D foi realizada por reação em cadeia da polimerase (PCR) a partir do DNA genômico. As distribuições genotípicas e alélicas foram comparadas com populações controle e de atletas pelos testes do qui-quadrado e exato de Fisher; todas as análises consideraram p ≤ 0,05. Resultados: As distribuições genotípicas e frequências alélicas do ACTN3 RR=46%, RX=38% e XX=16%; R=65% e X=35% e ACE I/D DD=47,7%, ID=34,3% e II=20%; D=62,9% e I=37,1% não diferiram da população controle, porém, quando comparadas com atletas de luta, constatou-se diferença significativa. Conclusão: Esses resultados sugerem uma associação dos genes ACTN3 R577X e ACE I/D aos lutadores de domínio de alto rendimento brasileiros.

RESUMEN Introducción: Al asociar genética y deporte, existe la posibilidad de identificar sujetos con mayor capacidad de adaptación a los entrenamientos con menores posibilidades de lesión. Objetivo: La investigación evaluó la distribución genotípica y alélica de los polimorfismos ACTN3 R577X y ACE I/D en luchadores brasileños de alto rendimiento de técnicas de agarre y golpeo. Métodos: Participaron en el estudio 37 atletas del sexo masculino, colocados de primer a tercer lugar en escenarios mundiales, divididos en dos grupos: agarre (23 luchadores, siendo 11 de judo, 4 de lucha grecorromana y 8 de jiu-jitsu; el promedio de edad fue de 27,3 ± 6,9 años) y golpeo (14 atletas con promedio de edad de 25,7 ± 4,4 años, siendo 6 de karate, 3 de muay-thai, 4 de taekwondo y 1 de boxeo). El genotipado de los polimorfismos de ACTN3 y ACE I/D se llevó a cabo por reacción en cadena de la polimerasa (PCR) a partir de ADN genómico. Las distribuciones genotípicas y alélicas fueron comparadas con poblaciones control y atletas por las pruebas del chi-cuadrado y exacta de Fisher; todos los análisis consideraron p ≤ 0,05. Resultados: Las distribuciones de los genotipos y frecuencias alélicas de ACTN3 RR=46%, RX=38% y XX=16%; R=65% y X=35% y ACE I/D DD=47,7%, ID=34,3% e II=20%; D=62,9% e I=37,1% no difirieron de la población control, sin embargo, cuando comparadas con los atletas de lucha, se constató diferencia significativa. Conclusión: Estos resultados sugieren una asociación de los genes ACTN3 R577X y ACE I/D a los luchadores brasileños de alto rendimiento de técnicas de agarre.

Trichomonas vaginalis α-Actinin 2 Modulates Host Immune Responses by Inducing Tolerogenic Dendritic Cells via IL-10 Production from Regulatory T Cells

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2, Tvα-actinin 2, has been used to diagnose trichomoniasis. This study was undertaken to examine the role of Tvα-actinin 2 as an antigenic molecule to induce immune responses from humans. Western blot analysis using anti-Tvα-actinin 2 antibodies indicated its presence in the secreted proteins of T. vaginalis. ELISA was employed to measure cytokine production by vaginal epithelial cells, prostate cells, mouse dendritic cells (DCs), or T cells stimulated with T. vaginalis or Tvα-actinin 2 protein. Both T. vaginalis and rTvα-actinin 2 induced cytokine production from epithelial cell lines, including IL-10. Moreover, CD4+CD25− regulatory T cells (Treg cells) incubated with rTvα-actinin 2-treated DCs produced high levels of IL-10. These data indicate that Tvα-actinin 2 modulates immune responses via IL-10 production by Treg cells.

The expression and clinical significance ofα-actinin-1 in prostate cancer and benign prostatic hyperplasia / 天津医药

Objective To investigate the expression and clinical significance of α-actinin-1 protein (ACTN1) in prostate cancer (PCa) and prostatic hyperplasia (BPH). Methods The clinical data of patients with PCa or BPH treated in our school affiliated hospital were collected between January 2007—October 2014, according to certain criteria. Immunohistochemistry method was used to detect the expression of ACTN1 in 30 samples of PCa and 30 samples of BPH tissues. Western blot assay was used to detect the relative expression of ACTN1 in 18 samples of PCa and 20 samples of BPH tissues in two groups. Results The result of immunohistochemistry showed that the positive expression rates of ACTN1 were 76.7%and 20%in PCa and BPH groups respectively. The difference was statistically significant (P 0.05). There were significant differences in ACTN1 levels between different Gleason score and T staging groups (P<0.05). Conclusion The expression ofα-actinin-1 is significantly higher in PCa tissues than that in BPH tissues. There is the relationship between expression of ACTN1, Gleason scores and T staging.

Polimorfismo del gen ACTN3 y ECA en seleccionados de gimnasia de Brasil y Japón / Gene polymorphism ACTN3 and ACE in selected gymnasts athletes in Brazil and Japan

En el rendimiento deportivo, el estudio de la genética ha cobrado un papel fundamental al momento de estudiar el rendimiento deportivo. El objetivo del estudio fue analizar la frecuencia de genotipo y alelo de a - actinina 3 (ACTN3) R577X y enzima convertidora de angiotensina (ECA) I/D polimorfismo en un grupo de gimnastas de Brasil y Japón. Se considero una muestra de 73 gimnastas (14 de Japón y 59 de Brasil), todos los sujetos firmaron de consentimiento informado. Para la obtención de ACTN3 y ECA se recogió una muestra la saliva y analizaron los genotipos mediante el empleo de cadena de la polimerasa en tiempo real a partir del iQ5 Thermal Cycler, BioRad. Los resultados muestran una prevalencia del genotipo RX ACTN3, prevalencia del alelo R, prevalencia del genotipo DI ECA y prevalencia de alelo D en el total del grupo. Se concluye que el predominio RX del ACTN3, prevalencia del alelo R y predominio del genotipo DI en ECA pueden ofrecer una ventaja genética en cuanto a niveles de fuerza y potencia muscular, posiblemente facilitando la práctica y el éxito competitivo en gimnastas.

In the field of competitive sport, genetics has gained a fundamental role in the study of sport performance. The aim of this study was to analyze the prevalence of polymorphisms R577X and insertion/deletion (I/D), occurring in a-actinin-3 (ACTN3) and angiotensin converting enzyme (ACE) genes, respectively, in Brazilian and Japanese gymnasts. A suitable non-probabilistic sample of 73 gymnasts (14 from Japan) was recruited and signed an informed consent. To measure ACTN3 and ECA saliva samples were obtained by means of real time polymerase chain reaction (iQ5 Thermal Cycler, BioRad). A high prevalence of RX ACTN3 genotype, R allele, ACE I/D genotype, and D allele were observed in Brazilian and Japanese gymnasts. In conclusion a high prevalence of RX ACTN3 genotype, R allele and ACE I/D genotype would allow a genetic advantage regarding muscle strength and power, possibly facilitating competitive success in gymnastics.

The effect of erythropoietin on myocardial cytoskeletal proteins after hypoxia/reoxygenation injury / 中华急诊医学杂志

Objective To observe the impact of hypoxia/reoxygenation on myocardial cytoskeletal proteins (α-actinin protein,tubulin protein,desmin protein) and to investigate EPO lessening the damage of myocardial cytoskeleton proteins in rats proved by culturing hypoxia/reoxygenation injured myocardial cells in presence of EPO.Methods The rat model of asphyxia-induced cardiac arrest was performed by turning-off the ventilator and clamping the endotracheal tube.After asphyxia for 8 minutes,CPR was carried out.A total of 24 rats were divided into normal group,ischemia/resuscitation (I/R) group and the EPO group (n =8).The model of myocardial dysfunction was determined 2 hours after restoration of spontaneous circulation (ROSC).The rats of EPO group were given EPO 5000 U/kg after ROSC.The rat heart specimens were collected.Actinin,Tubulin and Desmin protein were observed by SABC immunohistochemistry.The cultured cardiomyocytes were taken from neonatal rats and were divided into three groups:the normal group,the hypoxia/reoxygenation (H/R) group (hypoxia 10 h/reoxygenate 4h),the EPO group (hypoxia 10 h/reoxygenate 4 h,plus 10 U/mL EPO).The changes of tubulin and actinin in cultured cardiomyocytes were observe by Immunofluorescence.Results From immunohistochemistry,there were no significant difference in the optical density of actinin,tubulin and desmin among the normal,I/Rand EPO groups.After H/R injury,the structures of the actinin,tubulin protein were destroyed,the network structure of both protein were unclear in cultured myocardial cells.The grades of fluorescence intensity of actinin and tubulin in H/R group were significant lower than those in normal group,but there was no significant difference between H/R group and EPO group.Conclusions The damage of cytoskeleton during ischemia/reperfusion may be time-dependent.EPO has no beneficial effect on the cytoskeleton after I/R injury.

Molecular mechanism of dexamethasone affecting podocyte motility / 中华肾脏病杂志

Objective To investigate the potential roles of dexamethasone (Dex) in podocyte motility,and to explore the mechanism of modulating α-actinin-4,nephrin.Methods Podocytes were divided into three groups:Dex group [1 μmol/L Dex +50 mg/L puromycin aminonucleoside (PAN)],PAN group (50 mg/L) and normal control group.Scrape wound assay and Transwell migration assay were used to detect cell motility.Filtering ratio of podocytes was measured by FITC labeled BSA.Real-time PCR and Western blotting were used to examine the expressions of c-actinin-4 and nephrin.Results From the scrape wound assays,the ability of wound repair in podocytes of PAN group was significantly increased (P＜0.01),and the number of migrating cells in this group also rose (P＜0.01).Compared to PAN group,podocytes in Dex group did not enhance the motility after treatment with the same dose PAN (P＜0.01).Real-time PCR and Western blotting showed that Dex could significantly inhibit the up-regulated expression of α-actinin-4 and nephrin induced by PAN.Conclusions Dex can relieve the enhanced motility induced by PAN.Its mechanism may be associated with the modulation of the expressions of α-actinin-4 and nephrin.

Hepatitis B virus X protein induces expression of fibrosis-related factors in human hepatic stellate cells / 中国医师杂志

f fibrosis-related factors in LX-2cells were significantly increased after co-cultured with QSG7701-HBx cells, which proved that HBx could induce fibrogenesis in vitro.

The expression and significance of c-Ski in kidney of diabetic rats / 中国医师杂志

Objective To observe the expressions of c-Ski in renal tissue of diabetic nephropathy rats, and discuss its significance. Methods Wistar male rats were randomly assigned to 3 groups: Control group, diabetic group and treatment group. Rats were killed at the 16th week after experiment. Histopathologic changes,in renal tissue were observed and immunohistochemistry was performed to investigate the expression of TGF-β1, α-SMA and c-Ski. Results The expression of c-Ski had a negative correlation with TGF-β1 and α-SMA. Compared to diabetic group, the expression of c-Ski was significantly increased in treatment group. Conclusion c-Ski may be a protective factor of tu-bular epithelial-myofibroblast transformation.

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.

Study on molecular effect and interaction among podocyte molecules by gene knockdown / 中华肾脏病杂志

Objective To explore the molecular effect and interaction among nephrin, podocin, CD2AP and ?-actinin-4. Methods Firstly, the recombinant RNA interference (RNAi) plasmid-psiRNA-hH1GFPzeo, specifically targeting to the mRNA of nephrin, podocin, CD2AP or ?-actinin-4, was respectively tansfected into the mouse podocyte clone (MPC5) to each knockdown (KD) the expression of nephrin, podocin, CD2AP or ?-actinin-4. Molecular distributions were revealed by confocal microscopy, and the mRNA and protein expressions were detected with semi-quantitative RT-PCR and Western blotting. Results (1)In podocin KD group (siPod966 and siPod54), the mRNAs of podocin and nephrin were not detected, their protein decreased 92% and 79%, 82% and 67%, respectively. The mRNA and protein level of CD2AP increased 62% and 42%, 71% and 46%, respectively, whereas ?-actinin-4 did not change. In nephrin KD group (siNep492), the mRNA expression and protein level of nephrin were not detected, CD2AP increased 35% and 48%, respectively; and whereas podocin and ?-actinin-4 did not change. In CD2AP KD group (siCda744 and siCda21), the mRNA of expression CD2AP was not detected, and its protein level decreased 92% and 83%, the mRNA and protein of nephrin decreased 60% and 48%, 76% and 72%, respectively; podocin increased 38% and 22%, 56% and 44%, respectively; whereas ?-actinin-4 did not change. In ?-actinin-4 KD group (siAct1790 and siAct319), the mRNAs expression of ?-actinin-4 and nephrin decreased 69% and 58%, 64% and 49%, respectively; their protein level decreased 81% and 55%, 71% and 64%, respectively. However, the mRNAs of podocin and CD2AP increased 50% and 34%, 45% and 28%, respectively; and their protein level increased 64% and 46%, 65% and 42%, respectively. (2) With their expression change, the distributions of nephrin, podocin and CD2AP shifted evidently from the cell membrane surface to the nucleus circumference, whereas ?-actinin-4 showed no change, which was still localized in the cytoplasm and further extended to foot processes. Conclusion (1) Nephrin might more independently play a crucial role in the slit diaphragm complex. (2) Alpha-actinin-4 might interact direcdy or indirectly with nephrin, podocin and CD2AP. (3) The relationship among these podocyte molecules might not be spontaneous, either a single-directional or bi-directional reaction. (4) The normal localization of these podocyte molecules might depend on their normal expression quantity.

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.

The Relationship between Subcellular Localization of Actinin-4 and Cell Motility in Oral Squamous Cell Carcinoma / Oral Science International

To elucidate the mechanism of invasion of oral squamous cell carcinoma, we newly established two different cell lines with a high-motility phenotype (designated HI type) and low-motility phenotype (LI type) from CA-9-22, a human oral squamous cell carcinoma cell line, through cell invasion assay (Boyden chamber assay). When we examined the subcellular localization and protein expression of actinin-4 using these cell lines, although the growth curves were not significantly different between the HI type and LI type, more invasion was seen in the HI-type than in the LI-type on Boyden chamber assay (p<0.0001). Morphologically, a larger number of sharply extended cell processes and spindle formation were observed in the HI-type than in the LI-type, and actinin-4 was mainly distributed in these processes. Western analysis showed that the expression level of actinin-4 was almost equivalent between the HI and LI types. These findings suggest that subcellular localization of actinin-4 might be involved in cell motility and cancer invasion by regulating the actin cytoskeleton at the cell processes in oral squamous cell carcinoma.

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.