Desialización eritrocitaria con y sin exposición del antígeno T producida por Trichinella spiralis / Erythrocyte desialylation with and without T antigen exposure produced by Trichinella spiralis / Desialylation eritrocitaria com e sem exposição antigénio T produzida por Trichinella spiralis

El objetivo del trabajo fue estudiar la desialización de eritrocitos que desenmascaran el antígeno T y de los glóbulos que no lo exponen, por contacto con larvas recién nacidas (LRN) de T. spiralis. Se trabajó con 15 suspensiones eritrocitarias en medio enzimático, incubadas con LRN. Los GR control se incubaron con solución salina. Para analizar el efecto del parásito sobre la exposición del antígeno T, se aplicó el Método de Aglutinación anti-T con antígeno T y se estudió la agregación por los métodos de Polibrene y de Análisis Digital de Imágenes, determinando CexpST, CCA y Distribución de los agregados eritrocitarios (DAE). La media de los coeficientes en los eritrocitos que expusieron el antígeno T fueron CexpST=0,22±0,179 y coeficiente de células aisladas (CCA)=0,73±0,108 y en los que no 0,86±0,125 y 0,205±0,163 respectivamente. La DAE mostró que los GR que lo expusieron, presentaron marcada disminución de células aisladas y pronunciado aumento de grandes agregados en relación a los controles, mientras que los GR en los que no hubo exposición, la disminución de células aisladas con respecto a los controles fue menor y el aumento de agregados estuvo homogéneamente distribuido entre todas las categorías. La experiencia realizada concluye que los valores obtenidos de los coeficientes difieren significativamente en los GR que exponen el antígeno T y en los que no, por lo que estas técnicas podrían ser predictivas para detectar desenmascaramiento T.

The aim of this work was to study the desialylation of erythrocytes that expose the T antigen by contact with T. spiralis newborn larvae (NL) and of those that do not. Work was carried out with 15 red cell suspensions in enzymatic medium, which were incubated with NL. The respective Control RBC were incubated with saline solution. To analyze the effect of the parasite on the exposure of the T antigen, the Anti T- antigen T Agglutination Method was applied and the aggregation was studied by Polybrene and Digital Image Analysis Methods determining CexpTS, ICC and Distribution of Erythrocyte Aggregates (DEA). The mean of coefficients in the erythrocytes that exposed the T antigen were CexpTS= 0.22 ± 0.179 and ICC= 0.73 ± 0.108 and of those that did not it was 0.86±0.125 y 0.205±0,163 respectively. DEA showed that the RBCs that exposed it showed a marked decrease of isolated cells and a pronounced increase of large aggregates in relation to the Controls, while the RBCs in which there was no exposure, the decrease of isolated cells with respect to Controls was lower and the increase of aggregates was homogeneously distributed among all categories. The experience concludes that the obtained values of the coefficients differ significantly in the RBCs that expose the T antigen from those that do not, so these techniques could be predictive to detect T unmasking.

O objetivo do trabalho foi estudar a desialização de eritrócitos que desmarcaram o antígeno T e dos glóbulos que não o expõem, por contato com LRN de T. spiralis. Trabalhou-se com 15 suspensões de eritrócitos em meio enzimático, incubadas com LRN. Os GV controle foram incubados com solução salina. Para analisar o efeito do parasita sobre a exposição do antígeno T, foi aplicado o Método de Aglutinação anti T com antígeno T e se estudou a agregação pelos Métodos de Polibrene e de Análise Digital de Imagens, determinando CexpST, CCA e Distribuição dos agregados eritrocitários (DAE). A média dos coeficientes nos eritrócitos que expuseram o antígeno T foram CexpST= 0.22±0.179 e CCA= 0.73±0.108 e naqueles que não o expuseram 0.86±0.125 y 0.205±0,163 respectivamente. Foi demonstrado que os GV que o expuseram, apresentaram marcada diminuição de células isoladas e pronunciado aumento de grandes agregados em relação aos controles, enquanto que os GV nos quais não houve exposição, a diminuição de células isoladas com respeito aos controles foi menor e o aumento de agregados esteve homogeneamente distribuído entre todas as categorias. A experiência realizada conclui que os valores obtidos dos coeficientes diferem significativamente nos GV que expõem o antígeno T e nos que não, portanto estas técnicas poderiam ser preditivas para detectar desmascaramento T.

Inhibitory effect of Shiquandabu decoction on spontaneous len tumor of SV40 T antigen transgenic mice and its molecular mechanisms / 吉林大学学报(医学版)

Objective:To explore the inhibitory effect of Shiquandabu decoction on the spontaneous len tumor of the SV40 T antigen transgenic (TG) mice,and to clarify its molecular mechanism.Methods:The SV40 T antigen TG mice were randomly divided into control group (n=39) and drug treatment group (n=25).The mice in control group were fed normally,while the mice in drug treatment group were fed with Shiquandabu decoction at the 3rd week after birth,the survival time of mice was recorded.Three mice in control group and drug treatment group were randomly chosen to collect the blood from the tail vein and the amino acid levels were measured respectively 8 and 15 weeks after Shiquandabu decoction administration.Then the mice were sacrificed and the liver tissue wascollected.Gene chip hybridization was used to detect the differences in the expressions of ribosomal function related genes in liver tissue of the mice in two groups and the related signal pathway was explored.Results:The survival analysis demonstrated that the survival rate of TG mice in drug treatment was higher than that in control group (P＜0.05).Compared with control group,the serum levels of alanine,valine,leucine,isoleucine,threonine,methionine,proline,tyrosine,lysine,sarcosine,citrulline,ornithine and hydroxylysine of the mice in drug treatment group 8 weeks after administration of Shiquandabu decoction were increased (P＜0.05);and the serum levels of cystathionine,taurine,methylhistidine,anserine and ethanolamine were decreased (P＜0.05).Fifteen weeks after administration,compare with control group,the serum levels of threonine and citrulline of the mice in drug teeatment group were increased (P＜0.05),but the serum levels of other amino acids had no significant difference (P＞ 0.05).The canonical analysis showed that thirteen genes involved in ribosomal function from 9 083 genes in liver tissue in drug treatment group had the changes compared with control group (P＜ 0.05).Conclusion:Shiquandabu decoction can effectively prolong the lifetime of the TG mice by improving the levels of serum amino acids and promoting the liver ribosomal protein synthesis.

Inhibitory effect of Shiquandabu decoction on spontaneous len tumor of SV40 T antigen transgenic mice and its molecular mechanisms / 吉林大学学报(医学版)

Objective: To explore the inhibitory effect of Shiquandabu decoction on the spontaneous len tumor of the SV40 T antigen transgenic (TG) mice, and to clarify its molecular mechanism. Methods: The SV40 T antigen TG mice were randomly divided into control group (n=39) and drug treatment group (n=25). The mice in control group were fed normally, while the mice in drug treatment group were fed with Shiquandabu decoction at the 3rd week after birth, the survival time of mice was recorded. Three mice in control group and drug treatment group were randomly chosen to collect the blood from the tail vein and the amino acid levels were measured respectively 8 and 15 weeks after Shiquandabu decoction administration. Then the mice were sacrificed and the liver tissue was collected. Gene chip hybridization was used to detect the differences in the expressions of ribosomal function related genes in liver tissue of the mice in two groups and the related signal pathway was explored. Results: The survival analysis demonstrated that the survival rate of TG mice in drug treatment was higher than that in control group (P0. 05). The canonical analysis showed that thirteen genes involved in ribosomal function from 9 083 genes in liver tissue in drug treatment group had the changes compared with control group (P<0.05). Conclusion: Shiquandabu decoction can effectively prolong the lifetime of the TG mice by improving the levels of serum amino acids and promoting the liver ribosomal protein synthesis.

Exposición de criptoantígeno eritrocitario por larvas recién nacidas de Trichinella spiralis / Exposure of erythrocyte cryptic antigen by Trichinella spiralis newborn larvae / Exposição do cripto antígeno eritrocitário por larvas recém-nascidas de Trichinella spiralis

La desialización del eritrocito puede exponer determinantes antigénicas crípticas de la membrana, entre las cuales se encuentra el antígeno T. Se ha comunicado que las larvas recién nacidas de Trichinella spiralis (LRN), las cuales circulan por el torrente sanguíneo del hospedador, captan el ácido siálico eritrocitario. El objetivo de este trabajo fue estudiar in vitro la exposición del criptoantígeno T por la desialización producida por distintas concentraciones de LRN. Se trabajó con 30 suspensiones eritrocitarias en medio enzimático, que fueron incubadas en partes iguales con concentrados de LRN (GR Tratados) durante 2 horas a 37 ºC con agitación continua. Los respectivos GR Controles se pusieron en contacto con solución salina. El tratamiento de 10/30 suspensiones globulares se realizó con 500 LRN/mL, 10 con 300 LRN/mL y las últimas 10 con 150 LRN/mL. Se aplicó el Método de Aglutinación anti-T con antígeno T (en Placa y Tubo) enfrentando las suspensiones a suero de adulto y suero de cordón. El tratamiento de 9/10 de las suspensiones con 500 LRN/mL, el de 7/10 con 300 LRN/mL y el de 2/10 con 150 LRN/mL las expuso al criptoantígeno. Se concluye que en pacientes diabéticos, hipertensos o con otra patología que produzca disminución de ácido siálico eritrocitario y que cursen simultáneamente una infección por T. spiralis, en la etapa de circulación de larvas por el torrente sanguíneo podría producirse la activación T con la consecuente hemólisis, trombocitopenia y trombosis.

Erythrocyte desialylation can expose cryptic antigenic determinants of the membrane, among which is the T antigen. It has been reported that newborn larvae (NL), which circulate in the bloodstream of the host, capture erythrocyte sialic acid. The aim of this study was to study in vitro exposure of cryptic T antigen by the desialylation produced by different concentrations of NL. Work was carried out on 30 red cell suspensions in enzymatic medium, which were incubated in equal parts with NL concentrates (Treated RBC) for 2 hours at 37 ºC with continued agitation. The respective Control RBC was incubated with saline solution. Treatment of 10/30 globular suspensions was performed with 500 NL/mL, 10 with 300 NL/mL and the last 10 with 150 NL/mL. Anti T- antigen T Agglutination Tests (Plate and Tube) were made, facing the globular suspensions against adult and cord human sera. Treatment of 9/10 suspensions with 500 NL/mL, 7/10 with 300 NL/mL and 2/10 with 150 NL/mL exposed T antigen. It is concluded that in patients with diabetes, hypertension or other diseases with lower content of erythrocyte sialic acid and who simultaneously have a T. spiralis infection, T activation may occur in the stage of larvae circulating through the bloodstream, with consequent haemolysis, thrombocytopenia and thrombosis.

A dessialização do eritrócito pode expor determinantes antigênicos crípticos da membrana, entre os quais se encontra o antígeno T. Foi comunicado que as larvas recém-nascidas de Trichinella spiralis (LRN), as quais circulam na corrente sanguínea do hospedeiro, capturam o ácido siálico eritrocitário. O objetivo foi estudar in vitro a exposição do cripto antígeno T pela dessialização produzida por diferentes concentrações de LRN. Trabalhou-se com 30 suspensões eritrocitárias em meio enzimático, incubadas em partes iguais com concentrados de LRN (GV Tratados) durante 2 horas a 37 °C com agitação contínua. Os respectivos GV Controles entraram em contato com solução salina. Tratamento de 10/30 suspensões globulares foi realizado com 500 LRN/mL, 10 com 300 LRN/mL e as últimas 10 com 150 LRN/mL. Foi aplicado o Método de aglutinação anti T-antígeno T (em Placa e Tubo) enfrentando as suspensões a soro de adulto e soro de cordão. O tratamento de 9/10 suspensões com 500 NRL / mL, o de 7/10 com 300 LRN/mL e o de 2/10 com 150 LRN/mL expuseram o cripto antígeno. Conclui-se que em pacientes diabéticos, hipertensos ou com outra patologia que produza diminuição do ácido siálico eritrocitário, e que padeçam simultaneamente uma infecção por T. spiralis, na fase de circulação de larvas pela corrente sanguínea, poderia ocorrer a ativação T com a consequente hemólise, trombocitopenia e trombose.

Desenmascaramiento del antígeno T por larvas de Trichinella spiralis y Ascaris lumbricoides / T antigen unmasking by Trichinella spiralis and Ascaris lumbricoides larvae / Desmascaramento do antigeno T por larvas de Trichinella spiralis e Ascaris lumbricoid

La activación T es causada por cambios en la estructura de la membrana de los glóbulos rojos (GR), que producen la aglutinación de esas células transformadas con la mayoría de los sueros de adultos ABO compatible. La unión del antígeno T con su anticuerpo específico desencadena poliaglutinación, hemólisis, trombocitopenia y trombosis. El objetivo del trabajo fue estudiar el efecto de larvas de A. lumbricoides y T. spiralis sobre el desenmascaramiento del antígeno T eritrocitario. Se trabajó con 3 concentrados de larvas L1/ L2 de A. lumbricoides (CLAL), y 6 de Larvas Musculares de T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 y LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Se utilizaron GR Grupo O en medio enzimático. Los GR se incubaron con igual volumen de CLAL/ LM y los GR Controles con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando GR Tratados y Controles a sueros de adulto y de cordón. Los resultados mostraron que 2 de las 5 suspensiones de GR Tratados con CLAL3 y 1 de las 5 Tratadas con LM6, aglutinaron con suero de adulto, pero no con suero de cordón. Los GR incubados con los concentrados restantes y los Controles no aglutinaron con ninguno de los sueros. Se concluye que es importante continuar estos estudios para relacionar la activación T con las dosis infectantes en ascariosis y triquinosis, particularmente en patologías que cursen con déficit de ácido siálico.

T activation is caused by changes in the structure of red blood cells (RBC) membrane producing the agglutination of these transformed cells with the majority of adult ABO compatible sera. The union of T antigen with its specific antibody unleashes polyagglutination, haemolysis, thrombocytopenia, and thrombosis. The aim of the present work was to study the effect of A. lumbricoides and T. spiralis larvae on erythrocyte T antigen unmasking. Work was performed on 3 L1/ L2 larvae concentrates of A. lumbricoides (ALLC) and 6 muscle larvae concentrates of T. spiralis (ML): ALLC1 and ML1: 450-500 larvae/ mL; ALLC2 and ML2: 900-1,000 larvae/ mL; ALLC3 and ML3: 1,800-2,000 larvae/ mL; ML4: 3,000-3,500 larvae/ mL; ML5: 7,500-8,000 larvae/ mL; ML6: 20,000 larvae/ mL. Group O RBC in enzymatic medium were used. RBC were incubated with an equal volume of ALLC/ ML and the Controls with physiological solution for 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were made, facing Treated RBC and Controls against adult and cord human sera. The results showed that 2 of the 5 RBC suspensions treated with ALLC3 and 1 of the 5 RBC suspensions treated with LM6 agglutinated with serum from adult, but not cord serum. RBC incubated with the remaining concentrates and Control suspensions were not agglutinated with any of the sera. It can be concluded that it is important to continue these studies to correlate T activation with infective dose in ascariosis and trichinosis, particularly in pathologies that course with sialic acid deficiency.

Ativação T é causada por alterações da estrutura do membrana dos glóbulos vermelhos (GV), que produz a agregação dessas células transformadas com a maioria dos soros de adultos ABO compatível. A União de antígeno T com o anticorpo específico desencadeia poliaglutinação, hemólise, trombocitopenia e trombose. O objetivo do trabalho foi estudar o efeito de larvas da A. lumbricoides e T. spiralis sobre o desmascaramento do antígeno T eritrocitário. Trabalhou-se com 3 concentrados de larvas L1 / L2 da A. lumbricoides (CLAL) e 6 de larvas musculares da T. spiralis (LM): CLAL1 y LM1: 450-500 larvas/mL; CLAL2 LM2: 900-1000 larvas/mL; CLAL3 y LM3: 1800-2000 larvas/mL; LM4: 3000-3500 larvas/mL; LM5: 7500-8000 larvas/mL; LM6: 20.000 larvas/mL. Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina O sedimento globular foi incubado com igual volume de CLAL / LM e o de GR Controles com solução fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de Aglutinação em Placa e em Tubo, enfrentando os GV Tratados e Controles a soros humanos de adulto e de cordão. Se utilizaron GV Grupo O en medio enzimático. Os GV foram incubados com igual volume de CLAL/ LM e os GV Controles com solução fisiológica durante 120 minutos a 37 ºC. Realizaram-se Provas de Aglutinação em Placa e em Tubo, enfrentando GV Tratados e Controles a soros de adulto e de cordão. Os resultados mostraram que 2 das 5 suspensões de GV Tratadas com CLAL3 e 1 das 5 Tratadas com LM6, aglutinaram com soro de adulto, mas não com soro de cordão. Os GV incubados com as concentrações restantes e os Controles não aglutinaram com nenhum dos soros. Conclui-se que é importante continuar estes estudos para relacionar a ativação T com as doses infectantes em ascaríase e triquinose, particularmente em patologias que cursem com déficit de ácido siálico. controles, não aglutinaram com nenhum dos soros.

Exposición del antígeno T eritrocitario por acción de Ascaris lumbricoides / Exposure of erythrocyte T antigen by Ascaris lumbricoides action / Exposição do antígeno T eritrocitário por ação de Ascaris lumbricoides

La poliaglutinabilidad de los glóbulos rojos puede deberse al desenmascaramiento del antígeno T críptico debido a la acción de neuraminidasas microbianas que eliminan residuos terminales de ácido siálico en la membrana del hematíe. En experiencias preliminares se demostró que Ascaris lumbricoides capta ácido siálico del eritrocito y que las suspensiones globulares en medio enzimático, incubadas con este parásito, pierden totalmente la capacidad de agregación. El objetivo fue estudiar la exposición del antígeno T eritrocitario, por acción de A. lumbricoides sobre la carga aniónica de glóbulos rojos deficientes en ácido siálico. Se trabajó con 48 extractos de ejemplares adultos del parásito ([EA]) y un concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Se utilizaron eritrocitos Grupo O en medio enzimático de bromelina (GRB) y eritrocitos Control en medio salino (GRC). El sedimento globular de GRB se incubó con igual volumen de [EA]/ [CLAL] y el de GRC con solución fisiológica durante 120 minutos a 37 ºC. Se realizaron Pruebas de Aglutinación en Placa y en Tubo, enfrentando los GRB y GRC a sueros humanos de adulto y de cordón. Los resultados mostraron que el 33,33% de los GRB incubados con [EA] y el 66,67% de los GRB incubados con [CLAL] aglutinaron con suero de adulto, pero no con suero de cordón. Los GRC no aglutinaron con ninguno de los sueros. Es la primera vez que se comunica la exposición del antígeno T eritrocitario por acción de un parásito. La activación T podría producir autoaglutinación y hemólisis en el hombre adulto y representaría un factor de riesgo transfusional en la población infantil.

Red cell polyagglutination may be due to the unmasking of the cryptic T antigen by the action of microbial neuraminidases, which remove terminal sialic acid residues in the erythrocyte membrane. Previous experiences showed that Ascaris lumbricoides capture erythrocyte sialic acid and thatglobular suspensions in enzymatic medium, incubated with this parasite, completely lost the ability to aggregate. The aim of this work was to study the erythrocyte T antigen exposure by A. lumbricoides action on the anionic charge of sialic acid deficient red cells. Studies were done on 48 adult specimen parasite extracts ([AE]) and an L1/ L2 larvae concentrate ([ALLC]: 1300-1500 larvae/mL). Group O red cells in bromelain enzymatic medium (RCB) and Control erythrocytes in saline medium (RCC) were used. The RCB sediment was incubated with an equal volume of [AE]/ [ALLC] and the RCC sediment with physiological solution during 120 minutes at 37 ºC. Plate and Tube Agglutination Tests were performed, contrasting RCB and RCC with adult and cord human sera. The results showed that 33.33% of the RCB incubated with [AE] and 66.67% of the RCB incubated with [LCAL] agglutinated with serum from adult, but not cord serum. RCB were not agglutinated with any of the sera. It is the first time that erythrocyte T antigen exposure by a parasite action is communicated. T activation could produce autoagglutination and haemolysis in the adult and would represent a transfusion risk factor in the child population.

A poliaglutinabilidade dos glóbulos vermelhos pode ser resultado do desmascaramento do antígeno críptico T devido à ação de neuraminidases microbianas que eliminam resíduos terminais de ácido siálico na membrana da hemácia. Em experiências preliminares foi demonstrado que Ascaris lumbricoides capta ácido siálico do eritrócito e que as suspensões globulares em meio enzimático, incubadas com esta parasita, perdem totalmente a capacidade de agregação. O objetivo foi estudar a exposição do antígeno T eritrocitário, por ação de A. lumbricoides sobre a carga aniônica de glóbulos vermelhos deficientes em ácido siálico. Trabalhou-se com 48 extratos de exemplares adultos da parasita ([EA]) e um concentrado de larvas L1/ L2 ([CLAL]):1300-1500 larvas/mL). Foram utilizados eritrócitos Grupo O em meio enzimático de bromelina (GVB) e eritrócitos Controle em meio salino (GVC). O sedimento globular de GVB foi incubado com igual volume de [EA]/ [CLAL] e o de GVC com solução fisiológica durante 120 minutos a 37 ºC. Foram realizados Testes de Aglutinação em Placa e em Tubo, enfrentando os GVB e GVC a soros humanos de adulto e de cordão. Os resultados mostraram que 33,33% dos GVB incubados com [EA] e 66,67% dos GVB incubados com [CLAL] aglutinaram com soro de adulto, mas não com soro de cordão. Os GVC não aglutinaram com nenhum dos soros. É a primeira vez que se comunica a exposição do antígeno T eritrocitário por ação de uma parasita. A ativação T poderia produzir autoaglutinação e hemólise no homem adulto e representaria um fator de risco transfusional na população infantil.

The Construction and Expression Confirmation of JC Virus T Antigen Expression Plasmid in Gastric Mucosa / 中国医科大学学报

Objective To construct and confirm the JC virus(JCV) T antigen expression plasmid using mouse keratin 19 (K19) promoter specific for the gastric epithelial cells.Methods The Ndel site was mutated by FCR with Bell insertion at both sides.The DNA fragment digested by Bcl Ⅰ was ligated with the plasmid containing K19 promoter via Bam Ⅰ site.The DNA sequence was confirmed by restriction enzyme digestion and direct DNA sequencing.Cytokeratin 19 protein was examined to screen gastric carcinoma cell for transfection of K19-JCV T antigen expression plasmid by immunohistochemistry.The Western blot was employed to detect the JCV T antigen expression in the gastric carcinoma transfectant.Results K19-JCV T antigen expressing plasmid was successfully constructed.The ACS strongly expressed cytokeratin 19 protein and was selected for the transfection of K19-JCV T antigen expressing plasmid.JCV T antigen was positively expressed in the AGS transfectant.Conclusion The synonymous mutation and compatible ligation are useful in the plasmid construction.The methy lation of restriction enzyme should be considered.It is meaning for the transgenic animal model of gastric carcinoma to successfully construct the JC virus T antigen expression plasmid in gastric mucosa.

Effects of T-Synthase activity on the expressing of Tn/STn and T/ST antigens in gastric carcinoma tissues / 中华微生物学和免疫学杂志

acts was much lower than that in Tn-cell.Conclusion The expression of Tn,STn,T and ST antigen in gastric carcinoma tissues of different TNM stages is different.Tn antigen expression in tumor cells may be caused by the decrease of T-Synthase activity.

Construction of a SV40 Virus Large J Antigen Eukaryocyte Vector and Its Targeted Expression / 中华医院感染学杂志

OBJECTIVE To design and construct eukaryocyte expression vector of SV40 virus large T antigen and induce its targeted expression in eukaryocyte.METHODS SV40 large T gene which excised intron was cloned by SOE(splicing by overlapping extension) and digested with restricted enzymes EcoR Ⅰ and BamH Ⅰ.By the same methods,we got the digested product of pEGFP-N1.After that,the two fragments were ligated to form SV40(TEGFP) by Ligation Kit,and sequenced by TaKaRa ABI Prism Terminator Cycle Sequence Kit.The reconstructed vector was transfected into primary cultured human fibroblast using a Lipofectin transfection method.At 48 h(after) transfection,the expression of SV40T was detected with PCR and RT-PCR using specific primer of T gene.(RESULTS) The restricted enzymes digested and sequencing results showed that SV40 large T gene had cloned into pEGFP-N1 vector successfully.The genome DNA and total RNA were isolated from the positive cells.With these samples,the specific 288 bp fragment was amplified using PCR and RT-PCR.CONCLUSIONS The recombinant plasmid SV40TEGFP will be a stable and valuable molecular tool for human eukaryocyte study.

Distribution of T types and emm Genotypes of Streptococcus pyogenes in Jinju, 2004 / 대한진단검사의학회지

BACKGROUND: T antigens and emm genotypes are useful markers for epidemiologic investigation of Streptococcus pyogenes infections. Epidemiologic studies of S. pyogenes were performed on a large scale in Jinju. This was the third study being carried out in the same area over the past 10 years. METHODS: A total of 328 S. pyogenes were isolated from throat cultures obtained from asymptomatic schoolchildren in the Jinju area in 2004. T typing was performed by a slide agglutination, and emm genotyping by PCR and DNA sequencing. We compared the results of this study with those of the previous ones performed in 1995 and 2002. RESULTS: T5/27/44 were the most prevalent, accounting for 29.6% of all isolates; T12 and T6 were 13.4% and 10.7%, respectively, and T nontypeable was 3.4%. The emm44/61 type was the most prevalent accounting for 29.3%, and emm6 and emm1 were 11.6% and 9.8%, respectively. CONCLUSIONS: Newly recognized T5/27/44 and emm44/61 were the most prevalent, accounting for about 30% of all isolates, while T12 and emm12 were significantly decreased in 2004 compared to the results of previous years. This study demonstrated divergent features of S. pyogenes epidemiology over the past 10 years in the Jinju area.

Association of T Antigens with emm Genotypes of Group A Streptococci / 대한임상미생물학회지

BACKGROUND: T typing has been used as a screening test for epidemiologic studies of group A streptococci (GAS) infections or carriers, and M typing has been performed for virulence studies. However, M typing is difficult to perform in routine laboratories. Recently, genotyping of the emm gene, which encodes the M protein, has become available. We investigated which T antigen is closely associated with a certain emmgenotype. METHODS: GAS were collected from the children in Jinju who were asymptomatic carriers (N=349) or had acute pharyngitis (N=122) during the 3 year-period from 2002 through 2004. T typing was performed by a slide aggulutination, and emmgenotyping by PCR and DNA sequencing. RESULTS: More than 90% of T1, T3, T6, T12, T25, and T5/27/44 antigens were associated with emm1, emm3, emm6, emm12 and 22, emm75, and emm44/61 genotypes, respectively; however, other T antigens, such as T2, T4, T7, T11, and B3264, were not associated with any particular emm genotypes. CONCLUSION: Several T antigens are so closely associated with particular emm genotypes that one could predict emmgenotypes based on the result of T typing.

Immortalized hepatocytes established with a temperature-sensitive SV40 T antigen could be independent of T antigen / 대한내과학회지

BACKGROUND: Conditionally immortalized hepatocytes (CIH) can be cultured almost indefinitely at permissive temperatures (33 degrees C), but they undergo apoptosis at nonpermissive temperatures (37~39 degrees C) by the release of p53 through inactivation of T antigen, which is called T antigen dependency. This study was aimed at examining if T antigen-independent clones can develop from CIH. METHODS: CIH established with a temperature-sensitive T antigen (WA1) were cultured continuously at 39 degrees C. Three clones (W39B, W39C, and W39J) survived at this temperature and was subject to following analyses: the morphology, growth, apoptosis, the expression of T antigen and p53, telomerase, and the T antigen gene sequence. RESULTS: WA1 proliferated at 33 degrees C with the population doubling time of 30.8 +/- 1.7 hours, but they underwent cell death at 39 degrees C. However, T antigen-independent clones (W39B, W39C, and W39C) proliferated at 39 degrees C without undergoing apoptosis, suggesting they lost the temperature-sensitive characteristics. WA1 expressed the T antigen at 33 degrees C, but not at 39 degrees C, and this temperature-sensitive pattern was maintained in T antigen-independent clones. In p53 expression, however, T antigen-independent clones revealed a different pattern. p53 was detected even at 39 degrees C where it normally would not be detected. Telomerase was activated in all the analyzed cell lines. A temperature-sensitive point mutation at nucleotide position 3505 of the WA1 was retained in all T antigen-independent clones. CONCLUSION: CIH can lost temperature-sensitive characteristics and acquire an ability to proliferate at nonpermissive temperatures. These changes might be related to the change of p53 rather than the change of T antigen itself in these cell lines.

Endometrial Cell Culture: Isolation, Characterization, and Immortalization / 대한불임학회지

No abstract available.

Construction of retroviral vector inserted SV40 large T antigen gene and its expression in rat hepatocytes / 中国普通外科杂志

Objective To construct the retroviral vector inserted SV40 large T antigen gene and transfect it into rat hepatocytes, analyze the status of SV40 large T antigen gene expression in rat hepatocytes, and to establish important basis for clinical hepatocytes transplanation. Methods Retroviral vector inserted SV40 large T antigen gene was(constructed) by DNA recombinant techniques in vitro, then the combinant vector was determined with enzyme(digestion) and sequencing and was transfected into the PA317 cell lines by liposome mediation and screened(anti-G4)18 positive clones. The viral titer was determined with the NIH3T3. After transfected into separated and purified rat primary hepatocytes, the SV40 large T antigen gene expression was detected by PCR and immunohistochemical (methods). Results (1)SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation. (2)The titer of pseudovirion packed by PA317 cell lines was 1.3?10~6CFU/ml. (3)SV40LT antigen gene was(integrated) into rat primary hepatocytes and its expression in transfected cells at 24 hour was higher than 96 hour (P

Proliferation of Corneal Endothelial Cells by Delivery of SV40 Large T Antigen

PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells, we delivered liposome-protein complex into bovine corneal endothelial cells(BCEC). METHOD: SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the Ki-67 was detected by standard immunohistochemical methods. RESULT: The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation and Ki-67 expression. It was tested by time-course study. CONCLUSION: This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

SV40 large T antigen, a viral oncoprotein, is known to immortalize human diploid fibroblast by soaking up cellular RB and p53, but its frequency is extremely low. Additional genetic alteration is necessary for single-step immortalization. We attempted to find out what this alteration is by overexpressing cellular signal mediator genes; c-myc and cyclin D frequently amplified in many cancer cells. Overexpression of cyclin D did not affect the immortalization, but, overexpression of c-myc along with T antigen could immortalize normal human diploid fibroblast. Several cellular markers tested during immortalization process showed that p21, a cyclin-dependent kinase inhibitor and a marker of cellular senescence, disappeared in the life span-extended cells by T antigen and in the immortalized cells by c-myc. p21 was, however, elevated in the senescent cells and in the cells of crisis. Interestingly, p16 was upregulated whenever T antigen is overexpressed. Telomerase activity was also activated only in the immortalized cells. These results suggest that overexpression of c-myc contributes to immortalization of human diploid fibroblast by activating telomerase activity and suppressing p21 activity.

Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage

The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.

Transfection of SV 40 Large T Antigen into Corneal Endothelial Cells

The coeneal endothelium is essential for the maintenance of normal corneal hydration, thickness, and transparency. However, corneal endothelial cells are incapable of significant proliferation in vivo. As we age, the density of corneal endothelial (CEN) cells gradually decreases. The goal of our study is to explore the possibility of enhancing the proliferation of corneal endothelial cells by introduction of SV 40 large T antigen, a transforming protein. To this end, introduction of protein into CEN cells was assessed by liposome assisted beta-galactosidase transfection in vivo, ex vivo, and in vivo. In all cases, cells treated with liposome-protein complex have shown dramatic blue stain in beta-galactosidase activity staining. This result convinced us that we could artificially introduce a foreign protein into a cell. To ascertain where SV 40 large T antigen is localized in the cell, purified SV 40 large T antigen was transfected into the cells using liposome and its presence was determined immunohistochemically. We show that the liposome delivered SV 40 large is localized in the nucleus and mitotic figures which may suggest its functional activity.

Conditional immortalization of human fetal hepatocytes using an amphotropic retrovirus encoding temperature - sensitive SV40 large T antigen / 대한내과학회지

BACKGROUND: Human cells are almost never spontaneously immortalized in vitro. We tried to immortalize human fetal hepatocytes (h-FH) and evaluate the differentiational status and its change. METHODS: Hepatocytes were isolated from a liver fragment of 20 week old fetus and infected with amphotropic recombinant retrovirus containing a temperature- sensitive mutant of SV40 large T antigen and neomycin phosphotransferase gene. G418 resistant colonies were cloned and expanded. The cells which were able to divide more than 30 times were used to analyze various functions. RESULTS: The immortalization rate was 3.3 x 10-8 and two cell lines (C11, D21) were established. C11-60, C11-80, D21-30 and D21-60 (suffix number means the cell division counts) were evaluated. D21-30 was thougt to be imcompletely immortalized because a considerable portion of cells died during culture. The morphology was similar to that of epithelial cells except for D21-30 which looked like fibroblast. The cells grew rapidly at 33oC but stopped growing at 39oC. T antigen and p53 was expressed at 33oC but disappeared at 39oC, which suggest that T antigen binds to p53. Chromosomal changes were so marked that it was impossible to discriminate exact number. Albumin was secreted as about 1/10 as that of h-FH, but alpha-fetoprotein secretion stopped after immortalization. Telomerase was activated in both cell lines except for the incompletely immortalized cells D21-30. Telomere was elongated in competely immortalized cell lines, but it was rather shortened in D21-30 compared to that of h-FH. Macroscopic colonies did not develop in soft agar assay. CONCLUSIONS: We successfully immortalized human fetal hepatocytes. Although the cells are not likely to have oncogenicity, the functions are not so good, possibly due to marked chromosomal changes which are thought to occur before telomerase is activated during immortalization step.