The correlation between level of serum APPL1 and urinary albumin excretion rate in patients with type 2 diabetes mellitus / 中华内分泌代谢杂志

Objective To investigate the association of adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1(APPL1)with urinary albumin excretion rate in patients with type 2 diabetes mellitus (T2DM),and to explore the role of APPL1 in the development of diabetic kidney disease(DKD). Methods According to the urinary albumin/creatinine ratio(UACR),288 newly-diagnozed patients with T2DM were divided into normal albuminuria group(UACR<30 mg/g,n=116),microalbuminuria group(UACR 30 ~300 mg/g,n=95),and macroalbuminuria group(UACR>300 mg/g,n=77). 130 healthy subjects with matched sex and age were used as control group. Serum APPL1,tumor necrosis factor α(TNF-α),and adiponectin levels were measured by ELISA method. Results Serum APPL1 level in T2DM patients was significantly higher than that in control subjects (P<0.01), and increased with the rising of UACR. In patients with T2DM, serum APPL1 level was negatively correlated with estimated glomerular filtration rate(r=-0.246, P<0.01) while it was positively correlated with HbA1C, low density lipoprotein cholesterol, total cholesterol, triglycerides, insulin resistance index, serum creatinine,blood urea nitrogen, systolic blood pressure, TNF-α, and adiponectin(r=0. 119, 0. 167, 0. 209, 0.194,0.273,0.242,0.131,0.144,0.365, and 0.952, respectively, P<0.05 or P<0.01). Conclusion Serum APPL1 level in patients with T2DM was increased with the rising of UACR, suggesting that APPL1 may be involved in the development of DKD.

A Novel Method for Analysis of Tyrosine Phosphopeptides Based on a Centrifugal Enrichment Device / 分析化学

Protein tyrosine phosphorylation is an important post-translational modification and has become one of the most active areas in proteome research in recent years. Protein tyrosine phosphorylation plays a key regulatory role in the numerous signal transduction processes and in the occurrence & development of tumor. The study of tyrosine phosphorylation and activity of it corresponding tyrosine kinases is of great significance for the research of drug targets for cancer treatment. However, tyrosine phosphorylation only represents less than 0. 1% of the total cellular protein phosphorylation. Therefore, there is a great challenge to identify tyrosine phosphorylation in real complex samples. In this work, a new centrifugal device by combining application of titanium dioxide ( TiO2 ) and C18 reversed phase packing materials for phosphopeptide enrichment and separation was developed, which led to simplified procedure, reduced sample loss and minimized interference.This new centrifugal device was made of pipette tips, adapters and Eppendorf tubes. Sample loading, phosphopeptides enrichment, washing, eluting and separation were combined into this device and could be achieved by simple centrifugation. This device was capable of paralleled sample processing with improved analysis throughput. Tandem enrichment by anti-phosphotyrosine antibody resulted in efficient enrichment and large scale identification of phosphotyrosine peptides by mass spectrometry. As a result, 967 phosphotyrosine sites corresponding to 545 proteins were successfully identified from 5 mg of mouse liver proteins, demonstrating the robustness and potential of this new strategy.

A Novel Method for Analysis of Tyrosine Phosphopeptides Based on a Centrifugal Enrichment Device / 分析化学

Protein tyrosine phosphorylation is an important post-translational modification and has become one of the most active areas in proteome research in recent years. Protein tyrosine phosphorylation plays a key regulatory role in the numerous signal transduction processes and in the occurrence & development of tumor. The study of tyrosine phosphorylation and activity of it corresponding tyrosine kinases is of great significance for the research of drug targets for cancer treatment. However, tyrosine phosphorylation only represents less than 0. 1% of the total cellular protein phosphorylation. Therefore, there is a great challenge to identify tyrosine phosphorylation in real complex samples. In this work, a new centrifugal device by combining application of titanium dioxide ( TiO2 ) and C18 reversed phase packing materials for phosphopeptide enrichment and separation was developed, which led to simplified procedure, reduced sample loss and minimized interference.This new centrifugal device was made of pipette tips, adapters and Eppendorf tubes. Sample loading, phosphopeptides enrichment, washing, eluting and separation were combined into this device and could be achieved by simple centrifugation. This device was capable of paralleled sample processing with improved analysis throughput. Tandem enrichment by anti-phosphotyrosine antibody resulted in efficient enrichment and large scale identification of phosphotyrosine peptides by mass spectrometry. As a result, 967 phosphotyrosine sites corresponding to 545 proteins were successfully identified from 5 mg of mouse liver proteins, demonstrating the robustness and potential of this new strategy.

A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca2+, Mn2+ and Mg2+. Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca2+ than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Construction and verification of Ientiviral CRKL gene RNA interfering vector / 国际肿瘤学杂志

Objective To construct a lentiviral vector carrying CRKL gene RNA interfering( RNAi ).Methods The CRKL RNAi was selected and subcloned into the lentiviral vector,pGCL-GFP(including U6 promotor and green fluorescent protein),generating the lentiviral vector LV-shCRKL.The corrected CRKL was confirmed by endoenzyme digestion ,sequencing.Recombinant lentiviruses were produced by 293T cells following the eo-transfection of LV-shCRKL,with the packaging plasmids pHelper1.0 and pHelper2.0.The virus titer was detected by GFP expressions in 293T cells.Results Plasmid LV-shCRKL carried the correct sequence.The recombinant lentiviruse LV-shCRKL could be produced by co-transfection of LV-shCRKL to 293T cells.Conclusion The recombinant lentiviruse vector LV-shCRKL is constructed successful.

Screening of binding proteins interact with phosphotyrosine-interacting domain of DOC-2 by yeast two hybrid system / 中国肿瘤生物治疗杂志

Objective: To screen for proteins which can interact with phosphotyrosine-interacting domain (PID) of differentially expressed gene in human ovarian cancer cell line DOC-2 by yeast-two hybrid technique, so as to provide evidence for the signal pathway of DOC-2. Methods: The cDNA sequence of human DOC-2 gene was amplified and its PID domain (nDOC-2) was subcloned into the bait vector pGBKT7 of yeast two-hybrid system; the product was then used to screen an embryo brain cDNA library and the proteins interacting with nDOC-2 were identified. Quadrople dropout(QDO) medium and X-?-gal were used for selecting the positive clones. PCA was used to analyze the amplified sequence. After elimination of the false positive clones, the positive clones were sequenced and analyzed by bioinformatic methods. Results:Twenty-one candidate positive clones were obtained and 3 of them were plasmids encoding Homo sapiens partial mRNA for betaglycan (TBR III gene), Homo sapiens protocadherin gamma subfamily C 3 (PCDHGC3), and APLP1(amyloid beta precursor-like protein 1).Conclusion: The proteins obtained in this study may play important roles in the signal pathway of DOC-2, which provides a new orientation for DOC-2 gene therapy of ovarian cancers

Screening of DOC-2 interactive proteins and identification of the interaction between DOC-2 and TGF?Ⅲ receptor / 中国癌症杂志

Background and purpose:DOC-2 serves as one of the tumor suppressing genes in human ovarian cancer and plays a role in the process of cell growth and differentiation.This study was to investigate the role of DOC-2 in the TGF? signal pathway and verification of the interaction between DOC-2 and TGF?Ⅲ receptor.Methods:The bait vector was constructed by inserting the PID domain of DOC-2(nDOC-2)into yeast express vector pGBKT7.pGBKT7-nDOC2 was transformed into the yeast AH109 and confirmed to be expressed.After the human foetus brain cDNA library had been transformed,the positive clones was screened by both nutrition defect medium and X-?-gal.The putative positive clones were sequenced and analyzed to get the DOC-2 interactive proteins.Furthermore,after the DOC-2 cDNA and TGF?Ⅲ receptor cDNA had been co-transfected into the human ovarian cancer cell line HO-8910 together,the interaction between DOC-2 and TGF?Ⅲ receptor was investigated by immunoprecipitation and Western blot.Results:21 putative positive clones were picked after being screened and sequenced.Three of them were identified as Homo sapiens partial mRNA for betaglycan(TBR Ⅲ gene),homo sapiens protocadherin gamma subfamily C3(PCDHGC3)and APLP1(amyloid beta precursor-like protein 1).The analysis by immunoprecipitation and Western blot showed that the interaction between DOC-2 and TGF?Ⅲ receptor could form protein complex.Conclusions:The three encoding proteins might participate in the DOC-2 signal pathway.DOC-2 might play as an essential role in the TGF?signal pathway by interacting with TGF?Ⅲ receptor.

Cyclic nucleotide phosphodiesterase inhibition increases tyrosine phosphorylation and hyper motility in normal and pathological human spermatozoa

Our objective was to determine the effect of phosphodiesterase (PDE) inhibition on: 1) tyrosine phosphorylation of human spermatozoa at the tail level; and 2) sperm motion parameters and hyperactivated motility. The study was conducted with normozoospermic and asthenozoospermic samples incubated under in vitro capacitating conditions. The main outcome measures were computer-assisted sperm motion analysis and fluorescent immunodetection of phosphotyrosine-containing proteins. Pentoxifylline (PTX) was used as PDE inhibitor because of its wide use in the clinic. PTX-treatment significantly increased sperm velocity, hyperactivated motility and tyrosine-phosphorylation, both in normo and asthenozoospermic samples. Tyrosine-phosphorylation of tail proteins was highly conspicuous in both types of samples, showing no differential pattern after PTX-treatment. Normozoospermic samples treated with pentoxifylline showed an increase in the number of spermatozoa displaying hyperactivated movement and tyrosine-phosphorylation at the tail level. Preliminary data on asthenozoospermic samples exhibiting altered motion characteristics and defective phosphorylation of sperm-tail proteins showed that both defects can be concomitantly overcome by pentoxifylline treatment. Tyrosine-phosphorylation of sperm-tail proteins is underlying the enhancement of hyperactivated motility resulting from PDE inhibition by pentoxifylline.

Overexpression of Tyrosine Phosphatase Containing c-Src Homology SH-2 in Condyloma Acuminatum / 中华皮肤科杂志

Objective To explore the expression of tyrosine phosphatase containing c-Src homology SH2 (SHP-2) in condyloma acuminatum. Methods Phosphorylated tyrosine and SHP-2 were detected in skin lesions of 30 patients with condyloma acuminatum, tumor tissues of 13 cases of cervical cancer and normal foreskin tissues of 11 normal controls by SP immunohistochemical stain. Results In the foreskin tissue, the phosphorylated tyrosine and SHP-2 were mainly expressed on the nuclei of kerotinocytes adjacent to the basal layer, and the number of cells with positive expression was not so many. In condyloma acuminatum, the positive cells arranged in patches, phosphorylated tyrosine and SHP-2 distributed in the cytoplasm. In cervical cancer, the expression of phosphorylated tyrosine and SHP-2 were mainly in the cytoplasm, diffusely distributed, and not in the nuclei. The positive expression rates of phosphorylated tyrosine and SHP-2 in condyloma acuminatum and cervical cancer were 96.67% (29/30) and 100% (13/13), respectively. However the cytoplasm of normal foreskin showed negative expression(0/11). Conclusions There is an aberrant activation of phosphorylated tyrosine and SHP-2 in condyloma acuminatum and cervical cancer. SHP-2 may play an important role in the carcinogenesis.

Inhibition of PTK Pa t hway by Genistein in Diabetic Rat

The objective of this study is to determine if the elevated retinal levels of phosphotyrosine in the Zucker diabetic fatty rats can be reduced by systemic administration of genistein,a protein tyrosine kinase inhibitor. Sixteen male Zucker diabetic fatty rats were compared with sixteen malelean litter mates as a control. Rats were injected intraperitoneally with 13.6mg/kg B.Wt.of genistein (treated group) or dimethyl sulfoxide (DMSO)(non-treated group) twice two hours apart. Twenty four hours later, the retinas were processed for western blot analysis and immunohistochemical study. The Zucker diabetic fatty rats were found to have increased levels of phosphotyrosine as compared with the lean controls. Intraperitoneal administration of genistein strongly inhibited protein tyrosine phosphorylation in the diabetic rats.Genistein also inhibited the increase in PCNA,but this was less obvious than the decrease in phosphotyrosine. In the diabetic rats, the levels of activated PI3-K and MAPK were increased over control rats. Genistein reduced the levels of PI3-K and MAPK. Immunohistochemical studies of the retina showed decreased immunopositive reaction of PCNA in the retina of the diabetic rats treated with genistein. In conclusion, elevat-ed levels of phospotyrosine in the Zucker diabetic rats can be reduced by systemic administration of genistein. Genistein also reduced, although less, the levels of PCNA and activated forms of PI3-K and MAPK. Our results suggest that PTK inhibitor like genistein may find a role in the reduction of proliferative complications of diabetes mellitus.

Activation of Protein Tyrosine Kinase Pathway in Diabetic Rats

One mechanism of vascular damage in diabetic retinopathy is thought to result from the effect of hyperglycemia on the neural retina and microvasculature eventually causing the signs of capillary closure and clinical retinopathy. We reasoned that protein tyrosine kinase (PTK) pathways are abnormally activated in the retina prior to the onset of clinical changes. In this study, twelve Zucker diabetic fatty rats were compared with twelve male lean litter mates as control. The rats were examined for changes in the relative retinal levels of angiogenic growth factors, phosphotyrosine, PCNA, and tyrosine kinase pathway intermediates by western blot analysis. We found that the Zucker diabetic fatty rats have increased levels of the angiogenic growth factors, VEGF and PDGF. The levels of phosphotyrosine and PCNA, and phosphorylated intermediates, MAPK and PI3-K were also increased as compared with the lean control rats. These data indicate that the microvascular changes known to occur in this model are associated with elevation of VEGF and PDGF prior to the development of clinical apparent retinopathy. The elevations of VEGF and PDGF may explain in part the activation of the tyrosine kinase pathways, especially pathways that include MAPK and PI3K.

EFFECTS OF RECOMBINANT CILIARY NEUROTROPHIC FACTOR ON STAT3 EXPRESSION AND TYROSINE PHOSPHORYLATION IN THE INJURED NEURONS DURING PERIPHERAL NERVE REGENERATION / 解剖学报

Objective To study effects of recombinant ciliary neurotrophic factor(CNTF) on the changes of JAK\|STAT pathway and tyrosine phosphorylation in the injured neurons. Methods Sciatic nerve of rat was resected and sutured into silicone tube with local infusion of recombinant CNTF.The distribution and quantity of STAT3 and phosphotyrosine(PTyr) immunoreactivity in the neurons of L3\|L5 spinal anteriolateral nuclei and L5 spinal ganglion were observed and measured by immunohistochemical ABC method with computer image analysis. Results There was much STAT3 immunoreactivity in the neuron located in the nucleus of spinal anteriolateral nuclei.PTyr immunoreactivity showed higher in the cell membrane of spinal anteriolateral nuclei and in the cytoplasma and neucleus of spinal ganglion in CNTF group than that in SAL group.Conclusion\ The results suggest JAK\|STAT pathway in the injured motoneurons be activated and strengthened and tyrosine phosphorylation in the injured neurons be enhanced by recombinant CNTF.\;[