In resting platelets, the 17 ^th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 ^rd phenylalanine (PHE ⁵⁶³-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.
Filamins/metabolism* ; Platelet Glycoprotein GPIb-IX Complex/metabolism* ; Molecular Dynamics Simulation ; Ligands ; Protein Binding ; Blood Platelets/metabolism* ; von Willebrand Factor/metabolism*

OBJECTIVE: To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation. METHODS: Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot. RESULTS: The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2. CONCLUSION The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.
Codon, Nonsense ; Factor IX/metabolism* ; HeLa Cells ; Humans ; Mutation ; RNA Splicing ; RNA, Messenger/metabolism*

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

Antigens, Neoplasm ; metabolism ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Carcinoma, Renal Cell ; diagnosis ; metabolism ; pathology ; Humans ; Kidney Neoplasms ; diagnosis ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Grading ; Prognosis ; RNA-Binding Proteins ; metabolism ; Tumor Burden ; Vascular Endothelial Growth Factor A ; metabolism ; Von Hippel-Lindau Tumor Suppressor Protein ; metabolism

OBJECTIVETo study the expression of carbonic anhydrase IX (CAIX), PAX2 and PAX8 in different types of renal epithelial tumor and their association with clinicopathologic characteristics.

METHODSImmunohistochemical study by EnVision method was performed in order to assess the expression of CAIX, PAX2 and PAX8 in 155 cases of renal cell carcinoma and 4 cases of metastatic clear cell renal cell carcinoma (CCRCC). Ninety-six cases of non-neoplastic renal parenchymal tissue adjacent to CCRCC, 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma were used as controls.

RESULTSCAIX was commonly expressed in CCRCC (94.0%, 63/67), of which 77.8% (49/63) showed strong positivity. CAIX was focally positive in papillary renal cell carcinoma, collecting duct carcinoma and urothelial carcinoma of renal pelvis. It was negative in chromophobe renal cell carcinoma, oncocytoma and adjacent non-neoplastic renal tissue. CAIX was also strongly expressed in the 4 cases of metastatic CCRCC. Focal expression of CAIX was demonstrated in the 8 cases of clear cell hepatocellular carcinoma and 2 cases of clear cell hidradenoma. The expression of CAIX in CCRCC did not correlate with tumor grading, clinical staging and presence of distal metastasis. On the other hand, PAX2 showed positive expression in different types of renal epithelial tumor, clear cell hepatocellular carcinoma and clear cell hidradenoma in various degrees. In contrast, PAX8 was commonly expressed in all types of renal epithelial tumor, with the exception of urothelial carcinoma of renal pelvis. PAX8 was not expressed in clear cell hepatocellular carcinoma and clear cell hidradenoma. Regarding diagnosis of CCRCC, CAIX demonstrated high sensitivity and specificity. PAX2 showed high specificity but low sensitivity. PAX8 was sensitive and specific in the diagnosis of renal epithelial tumor.

CONCLUSIONSCAIX is a useful immunohistochemical marker with high specificity and sensitivity in distinguishing CCRCC from other types of renal epithelial tumor and clear cell tumors of non-renal origin. PAX2 is a marker with high sensitivity and low specificity for diagnosis of renal epithelial tumors. PAX8 is typically expressed in renal epithelial tumors. The combined detection of CAIX, PAX2 and PAX8 is useful in the diagnosis and differential diagnosis of renal epithelial tumors.

Adenoma, Oxyphilic ; metabolism ; pathology ; Antigens, Neoplasm ; metabolism ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Carcinoma, Renal Cell ; metabolism ; pathology ; Diagnosis, Differential ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Male ; PAX2 Transcription Factor ; metabolism ; PAX8 Transcription Factor ; Paired Box Transcription Factors ; metabolism

OBJECTIVETo explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha.

METHODSThe VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope.

RESULTSThe VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips.

CONCLUSIONSThe amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.

Amino Acid Sequence ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Platelet Adhesiveness ; Platelet Glycoprotein GPIb-IX Complex ; genetics ; metabolism ; von Willebrand Factor ; metabolism

This study was purposed to investigate the correlation of deep vein thrombosis (DVT) with C-reactive protein (CRP), fibrinogen (Fg), coagulation factor VIII (FVIII:C), coagulation factor IX (FIX:C) and to explore the effect of inflammation and coagulation as well as their interaction in DVT and its mechanism. 59 patients with DVT undergoing selective venous ultrasonography and 26 healthy individuals as controls were enrolled in this study. The plasma level of CRP was detected by immunoturbidimetry, FVIII:C, FIX:C levels were determined by a one-stage assay and fibrinogen level was measured by full-automatic biochemical apparatus. The results showed that the mean levels of plasma CRP, Fg, FVIII:C and FIX:C were significantly higher in deep vein thrombosis group than that in controls [CRP (2.67 +/- 0.91) vs (0.14 +/- 0.08) mg/dl; Fg (4.73 +/- 1.36) vs (2.79 +/- 0.66)g/L; FVIII:C (126.71 +/- 28.10) vs (81.35 +/- 20.77)%; FIX:C (81.01 +/- 23.60) vs (70.71 +/- 11.3)%] (p < 0.01), and the level of plasma CRP was strongly correlated with Fg, FVIII:C and FIX:C (r(s) = 0.432, 0.571 and 0.544, p < 0.01). It is concluded that the DVT and inflammation are closely related, increased level of plasma CRP may be a predictor of DVT. Increased plasma levels of Fg, FVIII:C and FIX:C all are important risk factors to DVT. Interaction between inflammation and coagulation promote the incidence of DVT, which may be one of DVT pathogenesis.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biomarkers ; blood ; Blood Coagulation ; C-Reactive Protein ; metabolism ; Case-Control Studies ; Factor IX ; metabolism ; Factor VIII ; metabolism ; Female ; Fibrinogen ; metabolism ; Humans ; Inflammation ; Male ; Middle Aged ; Venous Thrombosis ; blood ; etiology ; Young Adult

This study was aimed to investigate the effects of bortezomib on VEGF gene expression of endothelial cell line HMEC-1, and to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1 (HIF-1alpha) and expression intensity of Annexin A2, so as to analyze the possible mechanisms of the above expression of VEGF gene. Expression intensity of VEGF gene was determined by real-time quantitative PCR; the relative proliferation activity of cells was assayed by cell count kit CCK-8; the expression intensity of carbonic anhydrase IX (CA IX) gene was detected by RT-PCR; expression of Annexin A2 at gene and protein levels were determined by real-time quantitative PCR and Western blot respectively. The results showed that after being treated by bortezomib with 2.5, 5.0, 10 nmol/L for 12 hours, the expression intensity of VEGF gene of endothelial cell line HMEC-1 was as follows: 0.730 +/- 0.106, 0.673 +/- 0.153, 0.767 +/- 0.090 (as 1.0 was made in 0 nmol/L) (p < 0.05); the proliferation activity of cells was not significantly suppressed by bortezomib in 2.5, 5.0 nmol/L (p > 0.05), while that was significantly suppressed by bortezomib of 10 nmol/L (p = 0.024), The results from RT-PCR showed that expression intensity of CA IX gene was conspicuously down-regulated by bortezomib in different concentrations, which suggested that the transcriptional regulation activity of HIF-1alpha was inhibited by bortezomib. And down-regulated expression of Annexin A2 protein by bortezomib in different concentrations was confirmed by real-time quantitative PCR and Western blot. It is concluded that low doses of bortezomib has no significant inhibition effect on the activity of proteasome. Bortezomib may down-regulate the expression of VEGF gene of endothelial cell through regulating the activity of HIF-1alpha and the expression of Annexin A2.
Annexin A2 ; genetics ; metabolism ; Antigens, Neoplasm ; genetics ; metabolism ; Boronic Acids ; pharmacology ; Bortezomib ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; metabolism ; Cell Line ; Down-Regulation ; Endothelial Cells ; drug effects ; metabolism ; Gene Expression ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Pyrazines ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVE: To analyze the expression and relationship between hypoxia inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase IX (CA IX) in nasal polyps, and to explore the role of HIF-1alpha and CA IX in the pathogenesis of nasal polyps. METHOD: Twenty-eight tissues of nasal polyps and 12 tissues of normal inferior turbinate mucosa were collected in this study. And Immunohistochemistry method was used to detect the expression of HIF-1alpha and CA IX in the two groups. RESULT: The numbers and intensity of positive stained cells of HIF-1alpha and CA IX were higher in nasal polyps. IOD of HIF-1alphaa and CA IX (10(3)/HP) in the nasal polyps were respectively 21.76 +/- 3.52, 26.87 +/- 4.60 compared with 3.37 +/- 1.65, 3.25 +/- l.20 in the control group by image analysis. There were significant differences between the two groups (P < 0.05). Spearman correlation test showed that there was a close correlation between the HIF-1alpha and CA IX expression in nasal polyps (r = 0.820, P < 0.01). CONCLUSION The results showed that HIF-1alpha and CA IX were over expressed in nasal polyps, and there was a close correlation between them. Hypoxia was found in nasal polyps and it could become more severe under infection, inflammation and energy metabolic disorder which consumed a great deal of oxygen. HIF-1alpha was over expressed in nasal polyps, which activate and promote the expression for CA IX, in order to keep the normal pH of cells for adapting the hypoxic microenvironment and sustain persistent growth and hyperblastosis of cells in the hypoxic areas. At last the internal circle was forming, which accelerate the formation of nasal polyps.
Adolescent ; Adult ; Antigens, Neoplasm ; metabolism ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Case-Control Studies ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Middle Aged ; Nasal Polyps ; metabolism ; pathology ; Young Adult

CHO cells expressing human GPIb/IX and rabbit red blood cells coated with human von Willebrand factor (VWF) were adapted to our study on the binding probability and the detachment force of GPIb/IX and VWF. With the micropipette system, the two cells were impinged under a constant force for controlled time. When the cells were pulled apart, the deformation of RBC was recorded, and the binding score and detachment force of the proteins were determined. After the two cells were impinged into 0.5 microm for 30 s, the binding probability of the two cells carrying GPIb/IX and VWF was 15.0%. Via analyzing the deformation of red blood cells, we found out the distribution of rupture forces of cells with GPIb/IX and VWF. Therefore, we infer that the continuous distribution of the detachment force is due to the stochastic effect. The most probable value of the detachment force was 10 pN.
Animals ; Binding Sites ; Blood Platelets ; metabolism ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; Humans ; Platelet Activation ; physiology ; Platelet Glycoprotein GPIb-IX Complex ; chemistry ; metabolism ; Rabbits ; von Willebrand Factor ; chemistry ; metabolism