Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats / Alterações dos parâmetros bioquímicos séricos e fosforilação da tirosina em rim e fígado de ratos induzidos por estresse crônico

Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male Sprague-Dawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

Alterations of serum biochemical parameters and tyrosine phosphorylation in kidney and liver of chronic stress-induced rats

Abstract Chronic stress (CS) can contribute to dysfunction in several organs including liver and kidney. This study was performed to investigate the changes in serum biochemistry, histological structure, as well as in localization of tyrosine phosphorylated proteins (TyrPho) and Heat shock protein 70 (Hsp-70) in liver and kidney tissues of CS rats induced by two stressors (restrained and force swimming) for 60 consecutive days. Samples of blood, liver, and kidney were collected from adult male SpragueDawley rats in each group. Our results showed that serum biochemical parameters including corticosterone, blood sugar, urea nitrogen, creatinine, cholesterol, triglyceride, HDL-C, LDL-C, ALT, AST, alkaline phosphatase in CS group were significantly different from that in normal group in both liver and kidney tissues. Although histological structure was not changed. TyrPho expression was significantly increased in liver lysate but significantly decreased in kidney. Hsp-70 expression in liver increased whereas in kidney decreased. In conclusion, CS can induce changes in liver and kidney functions.

Resumo O estresse crônico (SC) pode contribuir para a disfunção em vários órgãos, incluindo fígado e rim. Este estudo foi realizado para investigar as alterações na bioquímica sérica, estrutura histológica, bem como na localização de proteínas tirosina fosforiladas (TyrPho) e proteína de choque térmico 70 (Hsp-70) em tecidos hepáticos e renais de ratos CS induzidas por dois estressores (restrito e natação forçada) por 60 dias consecutivos. Amostras de sangue, fígado e rim foram coletadas de ratos Sprague-Dawley machos adultos em cada grupo. Nossos resultados mostraram que os parâmetros bioquímicos séricos, incluindo corticosterona, glicemia, nitrogênio ureico, creatinina, colesterol, triglicerídeos, HDL-C, LDL-C, ALT, AST, fosfatase alcalina no grupo CS foram significativamente diferentes do grupo normal em ambos os fígados e tecidos renais. Embora a estrutura histológica não tenha sido alterada, a expressão de TyrPho aumentou significativamente no lisado hepático, mas diminuiu significativamente no rim. A expressão de Hsp-70 no fígado aumentou, enquanto que no rim diminuiu. Em conclusão, a CS pode induzir alterações nas funções hepáticas e renais.

How phosphorylation of RBCs band 3 protein affects storage quality of suspended red blood cells prepared from Tibetan high Hb population / 中国输血杂志

【Objective】 To investigate the quality changes of suspended red blood cells (SRBCs) prepared from the blood of Tibetan high Hb population, and explore the availability and safety of blood collected from Tibetan high Hb population. 【Methods】 The voluntary blood donors were grouped according to the Hb concentration at the initial screening: female blood donors from Tibet Autonomous Region (>3 500 m) with Hb≥190 g/L and male blood donors with Hb≥210 g/L were classified as plateau high hemoglobin group. A total of 13 male blood donors from Tibet Autonomous Region were recruited. And the female blood donors (n=13) with Hb(115~165) g/L and male blood donors (n=12) with Hb(120~185) g/L from Chengdu were classified as control group. Whole blood of 200 mL specification was centrifuged to remove the plasma, and MAP additive solution was added to prepare SRBCs, then SRBCs were divided into four aliquots (50 mL/bag and stored at 4℃. Parameters as blood routine, free Hb and hemolysis rate were measured aseptically at day 1, 14, 21, 35 of storage. And 10 mL SRBCs was used to extract membrane proteins for tyrosine phosphorylation detection of band 3 protein. 【Results】 The RBCs counts(×1012/L), hematocrit(%) and hemoglobin(g/L) of Tibetan high Hb group and control group were 6.76±0.95 vs 4.65±0.52, 63.3±6.8 vs 43.1±4.4 and 214.4±19.8 vs 143.2±16.9 (P<0.01). The erythrocyte deformability test on the day 1, 14, 21, 35 of storage showed that the deformability of SRBCs prepared from Tibetan high Hb group was significantly lower than that of the control group under shear stress of 3, 5.33, 9.49, 16.87, and 30 Pa, while the hemolysis rate of SRBCs prepared from the Tibetan high Hb group and the control group on the day 1, 14, 21, 35 were 0.050 2±0.040 2 vs 0.022 2±0.011 1, 0.055 4±0.043 vs 0.032 1±0.028 7, 0.061 2±0.025 9 vs 0.034 3±0.031 7 and 0.069 6±0.032 0 vs 0.044 0±0.033 3 (P<0.05). Western blotting showed that the cytoplasmic N-terminal Y21 of band 3 protein of SRBCs prepared from Tibetan high Hb group was highly phosphorylated. 【Conclusion】 The deformability of SRBCs prepared from the Tibetan high Hb group was significantly lower while the hemolysis rate of SRBCs was higher than that of the control group. The hemolysis rate of the SRBCs at the end of storage prepared from the Tibetan high Hb group meets the requirements of the national standard GB18469-2012(<0.8%). The increase of hemolysis rate of SRBCs prepared from the Tibetan high Hb group was closely related to the phosphorylation of band 3 protein.

Advances and application of enrichment technology in SH2 superbinder-based tyrosine phosphoproteomics / 生物工程学报

Tyrosine phosphorylation is one of the important protein phosphorylations in eukaryotes responsible for a variety of biological processes including cell signaling transduction, cell migration, and apoptosis. In the study of phosphoproteomics, due to the low stoichiometry of tyrosine phosphorylation (pTyr) proteins and sometimes limited initial sample, traditional phosphoproteomics enrichment technology is inefficient for the enrichment of pTyr peptides. Here, we review the substantial progress in tyrosine phosphoproteomics by preparation of limited amount sample and the newly introduced SH2 superbinder.

Application of tyrosine phosphoproteomics in biomedical research: a review / 生物工程学报

The enrichment of tyrosine phosphorylation sites plays an important role in the study of tyrosine phosphoproteomics and the commonly used enrichment methods are antibody affinity enrichment and SH2 superbinder enrichment. In addition, in order to achieve large-scale identification of tyrosine phosphorylation sites, biological mass spectrometry and bioinformatics have been applied in tyrosine phosphoproteomics. In-depth coverage research of tyrosine phosphoproteomics, revealing the dysregulated kinases in cancer process, may help us understanding the occurrence and development process of cancer. According to literature reports, three quarters of the oncogenes are tyrosine kinase genes. Therefore, tyrosine kinase inhibitors have received more and more attention as anticancer drugs. The application of tyrosine phosphoproteomics technology can identify tyrosine kinases related to cancer and other major diseases, so as to help finding tyrosine kinase inhibitors. In short, tyrosine phosphoproteomics technology can be applied in biomedical fields such as tyrosine kinase identification, tyrosine kinase inhibitor research, and tyrosine phosphorylation signal pathway research.

Changes of tyrosine phosphorylation in liver and kidney of polycystic ovarian rats induced by letrozole / Cambios de fosforilación de tirosina en hígado y riñón de ratas ováricas poliquísticas inducidas por letrozol

Letrozole (Letro) is a drug commonly used for breast cancer treatment since it can decrease estrogen level. In experimental animal, the Letro has been used to induce the polycystic ovarian syndrome (PCOS) model. Tyrosine phosphorylation (TyrPho) is an essential process in various biological functions both normal and abnormal conditions especially reproduction. Although some side effects of Letro are reported, the alterations of TyrPho responsible for liver and kidney functions have never been demonstrated. In this study, the blood serum, liver, and kidney of control and PCOS rats induced with Letro (orally, 1 mg/ KgBW) for consecutive 21 days were used to determine the serum biochemical components and to investigate the TyrPho expression using western blot analysis. Histopathology of such tissues was observed by Masson's trichrome staining. The results showed that Letro did not affect histological structures but significantly increased the serum levels of urea nitrogen, cholesterol, triglyceride, HDL, LDL, ALT, AST, and alkaline phosphatase. Additionally, the TyrPho protein expressions of 32 and 27 kDas in liver and of 55 and 43 kDas in kidney were increased while of a kidney 26 kDa was decreased as compared to those of control. In conclusion, this recent study indicated that the changes of TyrPho proteins in liver and kidney induced with Letro associated with their functions by alteration of serum biochemical levels.

El letrozol (Letro) es un medicamento utilizado comúnmente para el tratamiento del cáncer de mama, debido a que puede disminuir el nivel de estrógeno. En animales de experimentación, el Letro se ha utilizado para inducir el modelo de síndrome de ovario poliquístico (PCOS). La fosforilación de tirosina (TyrPho) es un proceso esencial en diversas funciones biológicas, tanto en condiciones normales como anormales, especialmente en la reproducción. A pesar de informes que indican algunos efectos secundarios de Letro, no se han demostrado las alteraciones de TyrPho responsables de las funciones hepáticas y renales. En este estudio, el suero sanguíneo, el hígado y el riñón control y las ratas PCOS inducidas con Letro (por vía oral, 1 mg / KgBW) durante 21 días consecutivos se usaron para determinar los componentes bioquímicos del suero y para investigar la expresión de TyrPho usando análisis de transferencia Western. La histopatología de los tejidos se observó mediante la tinción tricrómica de Masson. Los resultados mostraron que Letro no afectó las estructuras histológicas, pero aumentó significativamente los niveles séricos de urea, colesterol, triglicéridos, HDL, LDL, ALT, AST y fosfatasa alcalina. Además, las expresiones de la proteína TyrPho de 32 y 27 kDas en el hígado y de 55 y 43 kDas en el riñón aumentaron mientras que en un riñón disminuyeron 26 kDa en comparación con el control. En conclusión, este estudio indicó que los cambios de las proteínas TyrPho en el hígado y los riñones inducidos con Letro se asociaron con sus funciones mediante la alteración de los niveles bioquímicos en suero.

The alterations of microvasculature, tyrosine phosphorylation, and lipid peroxidation in kidney of rats treated with valproic acid / Alteraciones de la microvasculatura, fosforilación de tirosina y peroxidación lipídica en riñones de ratas tratadas con ácido valproico

SUMMARY: Valproic acid (VPA), an antiepileptic drug, has been demonstrated to damage histology and to change tyrosine phosphorylation patterns with increased oxidative stress in perirenal tissues. This study aimed to investigate the effect of VPA on microstructure, tyrosine phosphorylation, and lipid peroxidation of rat kidney. Adult male rats were divided into control and VPA-treated groups intraperitoneally injected with normal saline and VPA 500 mg/kgBW for 10 consecutive days, respectively (n = 7 each). The blood serum was examined for biochemical levels. The kidney tissues were routinely processed for histological observation. Total proteins from kidney were extracted to assay the malondialdehyde (MDA) levels and phosphorylation expression. The results showed that VPA significantly decreased blood glucose levels while tend to increase urea nitrogen and creatinine. MDA levels in VPA group were significantly higher that of control. Renal cortex of VPA-treated animals revealed vasodilatations. Although the ratio of a renal phosphorylated 72 kDa protein/ beta actin expression seemed to be not different in both groups, VPA significantly decreased the intensity of beta actin. In conclusion, VPA dilates renal microvasculature with increasing of MDA but suppresses the actin expression.

RESUMEN: Se ha demostrado que el ácido valproico (AVP), un fármaco antiepiléptico, daña la histología y cambia los patrones de fosforilación de la tirosina con el aumento del estrés oxidativo en los tejidos perirrenales. Este estudio tuvo como objetivo investigar el efecto del AVP en la microestructura, la fosforilación de la tirosina y la peroxidación lipídica del riñón de rata. Se dividieron ratas macho adultas en grupos control y tratados con AVP. Durante 10 días consecutivos fueron inyectadas por vía intraperitoneal con solución salina normal y 500 mg / kg de PC respectivamente (n = 7 cada uno). Se analizó el suero sanguíneo para determinar los niveles bioquímicos. Los tejidos renales se procesaron de forma rutinaria para la observación histológica. Las proteínas totales del riñón se extrajeron para analizar los niveles de malondialdehído (MDA) y la expresión de la fosforilación. Los resultados mostraron que el AVP disminuyó significativamente los niveles de glucosa en la sangre, mientras que tienden a aumentar el nitrógeno ureico y la creatinina. Los niveles de MDA en el grupo de AVP fueron significativamente más altos que los del control. La corteza renal de los animales tratados con AVP reveló vasodilataciones. Aunque la proporción de una expresión de proteína / actina de 72 kDa fosforilada renal no parece ser diferente en ambos grupos, el AVP disminuyó significativamente la intensidad de la actina beta. En conclusión, el AVP dilata la microvasculatura renal al aumentar el MDA, pero suprime la expresión de actina.

Tyrosine phosphorylation of Kv1. 5 is upregulated in intrauterine growth retardation rats with exaggerated pulmonary hypertension

Intrauterine growth retardation (IUGR) is associated with the development of adult-onset diseases, including pulmonary hypertension. However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular dysfunction later in life is not fully understood. Here, we investigated the role of tyrosine phosphorylation of voltage-gated potassium channel 1.5 (Kv1.5) in this prenatal event that results in exaggerated adult vascular dysfunction. A rat model of chronic hypoxia (2 weeks of hypoxia at 12 weeks old) following IUGR was used to investigate the physiological and structural effect of intrauterine malnutrition on the pulmonary artery by evaluating pulmonary artery systolic pressure and vascular diameter in male rats. Kv1.5 expression and tyrosine phosphorylation in pulmonary artery smooth muscle cells (PASMCs) were determined. We found that IUGR increased mean pulmonary artery pressure and resulted in thicker pulmonary artery smooth muscle layer in 14-week-old rats after 2 weeks of hypoxia, while no difference was observed in normoxia groups. In the PASMCs of IUGR-hypoxia rats, Kv1.5 mRNA and protein expression decreased while that of tyrosine-phosphorylated Kv1.5 significantly increased. These results demonstrate that IUGR leads to exaggerated chronic hypoxia pulmonary arterial hypertension (CH-PAH) in association with decreased Kv1.5 expression in PASMCs. This phenomenon may be mediated by increased tyrosine phosphorylation of Kv1.5 in PASMCs and it provides new insight into the prevention and treatment of IUGR-related CH-PAH.

Research status of src-homology 2 domain-containing protein tyrosine phosphatase 1 in liver diseases / 第二军医大学学报

Protein tyrosine phosphorylation is ubiquitously found in eukaryotic cells, and it is a fundamental regulatory mechanism to be involved in the regulation of cell proliferation, apoptosis, differentiation and migration. Dysregulation of protein tyrosine phosphorylation can influence various liver diseases, such as liver inflammation, liver fibrosis and liver cancer. The src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1), a nonreceptor type protein tyrosine phosphatase, is a key regulator of maintaining protein tyrosine phosphorylation level in vivo. Recently, great progress in the research of SHP-1 in liver diseases has been made, and this review summed up the advances of SHP-1 in liver diseases.

Oviductal epithelial cells selected boar sperm according to their functional characteristics / 亚洲男科学杂志(英文版)

The interaction of oviductal epithelial cells (OECs) with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization (IVF), tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF: (i) W30 group: spermatozoa were incubated with oocytes in the absence of OEC; (ii) NB group: after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and (iii) B group: after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates (NB: 19.6%, B: 94.9%, and W30: 62.9%; P < 0.001) and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups (NB: 58.7%, B: 2.5%, and W30: 4.5%; P < 0.01). A similar trend was observed in phosphatidylserine translocation (NB: 93.7%, B: 5.7%, and W30: 44.2%; P < 0.01). These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

Verificación de los valores de referencia del estudio del semen según OMS 2010 en Buenos Aires / Verification of reference values for semen analysis according to WHO 2010 in Buenos Aires / Verificação dos valores de referência da análise do sêmen de acordo com a OMS 2010 em Buenos Aires

La Organización Mundial de la Salud en su edición 2010 ha publicado valores de referencia para los parámetros seminales. Sin embargo, los laboratorios deben verificarlos en su población para transferirlos a su práctica clínica. Para cumplimentar este requerimiento se propuso verificar los intervalos de referencia OMS 2010 en la población de la provincia y Ciudad Autónoma de Buenos Aires. También se determinó el rango de resultados para: número total de espermatozoides móviles progresivos y morfología normal en el eyaculado, parámetros cinéticos y pruebas funcionales. El proceso de verificación fue llevado a cabo de acuerdo a la guía C28-A2 CLSI (Clinical and Laboratory Standards Institute). Mediante difusión se convocó a hombres (n:20) argentinos residentes en Buenos Aires, con fertilidad probada en los últimos 12 meses. Se excluyeron individuos con patología andrológica. Las muestras se procesaron de acuerdo con los criterios OMS 2010. De los resultados se establece que los intervalos de referencia publicados en la última versión han sido verificados, con excepción del volumen seminal. Los rangos de valores obtenidos para los parámetros de movilidad progresiva y morfología en número totales, como también de los parámetros cinéticos y de las pruebas funcionales contribuirán a establecer futuros valores de referencia.

WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate,nematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.

Organização Mundial da Saúde, em sua edição de 2010, tem publicado valores de referência para os parâmetros seminais. No entanto, os laboratórios devem verificá-los em sua população para serem transferidos a sua prática clínica. Para cumprir com esse requisito, decidiu-se verificar os intervalos de referência da OMS 2010, na população da província e da cidade de Buenos Aires. Também foram definidos intervalos de resultados para o número total de espermatozoides móveis progressivos e para a morfologia normal no ejaculado, os parâmetros cinéticos e os testes funcionáis. O processo de verificação foi realizado de acordo com o guia CLSI C28 A2 (Clinical and Laboratory Standards Institute). Por meio de uma convocação, (N 20) homens argentinos residentes em Buenos Aires, com comprovada fertilidade nos últimos 12 meses foram convidados. Indivíduos com patologia andrológica foram excluídos. As amostras foram processadas de acordo com os critérios da OMS de 2010. A partir dos resultados estabeleceu-se que os intervalos de referência publicados na versão mais recente foram verificados, com exceção do volume seminal. Os intervalos de valores obtidos para os parâmetros de motilidade progressiva e morfologia em número total, bem como os parâmetros cinéticos e os dos testes funcionais ajudarão a estabelecer valores de referência futura.

The Short Term Effect of Insulin on Proinsulin Gene Expression of HIT-T15 Insulinnoma Cells / 天津医药

Objective To investigate the short term effect of insulin on proinsulin gene expression of HIT-T15 insu-linnoma cells(pancreatic isletβ-cell). Methods The HIT-T15 cells were randomly divided into four groups.Blank Con-trol Group (LG):complete medium contain 1.4 mmol/L glucose. Control group (LGC):co-cultured nifedipine with medium in order to restain endogenous insulin release. Experimental group (LINS or HINS) add 0.5 U/L insulin or 5 U/L insulin on top of LGC. After being stimulated for 0, 30, 60, 90, 120 mins, proinsulin (PI) mRNA level were assessed by semi-quantitative RT-PCR. Insulin receptor substrate1 (IRS1) tyrosine phosphorylation was detected by immunocytochemistry. Results (1) Expression of PI was up regulated by both LINS and HINS, and peak at 60 mins. (2) After stimulation for 30 mins, the level of IRS1 tyrosine phosphorylation in the experimental group was significantly higher than control group, and the peak time be-tween LINS and HINS was different. (3) Between group of LG and LGC, the expression of PI mRNA and IRS1 tyrosine phos-phorylation show no difference. Conclusion Short term exogenous insulin stimulation can promote expression of proinsulin genes,which is concentration dependent. The expression and regulation of PI were related with IRS1 signal transduction.

Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation

It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the affinity of ER414. We obtained a 3D model of ER414 to identify the amino acids in the CDRs that needed to be mutated. Clones were selected from the phage library with randomized amino acids in the CDRs and substitution of amino acids in the HCDR3 and LCDR1 of ER414 led to improved affinity. A clone, H3-14, with a ~20-fold increased affinity, was selected from the HCDR3 randomized library. Then three clones, ER2, ER78 and ER79, were selected from the LCDR1 randomized library based on the H3-14 but did not show further increased affinities compared to that of H3-14. Of the three, ER2 was chosen for further characterization due to its better expression than others. We successfully performed affinity maturation of ER414 and obtained antibodies with a similar affinity as cetuximab. And antibody from an affinity maturation inhibits the EGF-mediated tyrosine phosphorylation of EGFR in a manner similar to cetuximab.

A transient increase in total head phosphotyrosine levels is observed upon the emergence of Aedes aegypti from the pupal stage

Phosphorylation and dephosphorylation of protein tyrosine residues constitutes a major biochemical regulatory mechanism for the cell. We report a transient increase in the total tyrosine phosphorylation of the Aedes aegypti head during the first days after emergence from the pupal stage. This correlates with an initial reduction in total head protein tyrosine phosphatase (PTP) activity. Similarly, phosphotyrosine (pTyr)-containing bands are seen in extracts prepared from both male and female heads and are spread among a variety of structures including the antennae, proboscis and the maxillary palps combined with the proboscis. Also, mosquitoes treated with sodium orthovanadate, a classical PTP inhibitor, show reduced blood-feeding activity and higher head tyrosine phosphorylation levels. These results suggest that pTyr-mediated signalling pathways may play a role in the initial days following the emergence of the adult mosquito from the pupal stage.

Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome

The population of India harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.

Effects of Valsartan on Ventricular Hypertrophy and Expression of Proline-rich Tyrosine Kinase 2 in Myocardium of Renovascular Hypertensive Rats / 中华高血压杂志

Backgroud and Objective Proline-rich tyrosine kinase2(Pyk2) is a Ca~(2+) sensitive,non-receptor tyrosine protein kinase.Previous reports showed Pyk2 involved in development of left ventrieular hypertrophy. The present paper aimed to study the effects of valsartan on ventricular hypertrophy and its effect on the expression of Pyk2 in myocardium in renovascular hypertensive rats(RHR).Methods Two-kidney and one-clip(2K1C) renal hypertensive model was established in Sprague-Dawley rats by chronic partial occlusion of left renal artery,and ran- domized to receive valsartan (30 mg/kg?d) or without treatment for 4 or 8 weeks.Left ventricular mass to body mass ratio was measured.Pyk2 protein expression and phosphorylation was detected by Western blotting.Results Blood pressure,left ventricular mass to body mass ratio,Pyk2 activity in myocardium of RHR were increased gradu- ally.Valsartan reduced BP and prevent myocardial hypertrophy(P

The Localization and Expression of Tyrosine Phosphorylated Proteins During In Vitro Capacitation of Guinea Pig Sperm / 中国生物工程杂志

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

Involvement of MAP Kinases in Apoptosis of Macrophage Treated with Trichomonas vaginalis

A primitive protozoan parasite Trichomonas vaginalis selectively activates the signal transduction pathways in macrophages (RAW264.7). This study evaluated the correlation of these signaling pathways and T. vaginalis-induced cell apoptosis. In macrophages infected with T. vaginalis, apoptosis was assessed on the basis of DNA fragmentation on agarose gel electrophoresis. Infection of macrophages with T. vaginalis induced tyrosine phosphorylation of several proteins. Infected cells with T. vaginalis were shown to associate with phosphorylation of the extracellular signal-regulated (ERK) 1/2 kinase, p38, c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases on Western blot analysis. The present finding also demonstrated a link between the ERK1/2, JNK and p38 apoptotic pathways that was modulated by T. vaginalis infection.

Fyn Tyrosine Kinase-mediated Tyrosine Phosphorylation of Roundabout (Robo), the Slit Receptor / 대한해부학회지

In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.