OBJECTIVETo explore the relationship between hypoxia and the apoptosis of corpus cavernosum smooth muscle cells (CCSMC) in SD rats.

METHODSCCSMCs were cultured in vitro and identified by immunohistochemistry, and then underwent hypoxia interference at the concentration of 1% O2 for 12, 24, 48 and 72 hours, with normal oxygen concentration as the control. Flow cytometry was used to determine the cycles and apoptosis of the cells.

RESULTSThe cultured CCSMCs grew well, positive for anti-smooth muscle alpha-actin monoclonal antibody immunohistochemical staining. Flow cytometry showed that the number of CCSMCs in G0/G1 was gradually increased within 48 hours and then decreased, just opposite to the proportion of the S phase cells. But no regular change was found in the proportion of the cells in the G2/M phase.

CONCLUSIONHypoxia promotes the apoptosis of CCSMCs in a time-dependent manner, to the maximum at 48 hours, and then cell lysis may occur, but with no further apoptosis.

Animals ; Apoptosis ; Cell Hypoxia ; Cells, Cultured ; Male ; Muscle, Smooth, Vascular ; cytology ; Penis ; pathology ; Rats ; Rats, Sprague-Dawley

Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG. The recombinant fragment comprising residues 718-905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.
ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Escherichia coli ; genetics ; Humans ; Peptide Fragments ; biosynthesis ; genetics ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; metabolism ; Recombinant Proteins ; biosynthesis ; isolation & purification ; von Willebrand Factor ; biosynthesis ; chemistry ; genetics

BACKGROUNDThrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.

METHODSThe plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover, the serum vWF-cp activities were compared between the patients with TTP and those with tumors.

RESULTSThe coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79 +/- 9.17)% (n = 30) and (79.47 +/- 10.78)% (n = 53), respectively, while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83 +/- 19.98)%, P < 0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased (P < 0.03 and P < 0.001, respectively), they were relatively high in comparison with that of TTP patients (P < 0.001).

CONCLUSIONMeasurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.

ADAM Proteins ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Metalloendopeptidases ; blood ; deficiency ; Middle Aged ; Neoplasms ; enzymology ; Purpura, Thrombotic Thrombocytopenic ; enzymology

Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; CD2 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; Cyclophosphamide ; therapeutic use ; Doxorubicin ; therapeutic use ; Follow-Up Studies ; Humans ; Lymphoma, Large-Cell, Anaplastic ; drug therapy ; metabolism ; pathology ; Lymphoma, T-Cell, Cutaneous ; drug therapy ; metabolism ; pathology ; Male ; Neoplasms, Second Primary ; drug therapy ; metabolism ; pathology ; Poly(A)-Binding Proteins ; metabolism ; Prednisone ; therapeutic use ; Skin Neoplasms ; drug therapy ; metabolism ; pathology ; T-Cell Intracellular Antigen-1 ; Vincristine ; therapeutic use

ADAM Proteins ; biosynthesis ; genetics ; ADAMTS13 Protein ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Cell Line ; Female ; Humans ; Liver Neoplasms ; metabolism ; Middle Aged ; von Willebrand Factor ; biosynthesis ; genetics

The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.
ADAM Proteins ; biosynthesis ; genetics ; immunology ; ADAMTS13 Protein ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding Sites ; genetics ; Blotting, Western ; Epitopes ; immunology ; Escherichia coli ; genetics ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; von Willebrand Factor ; biosynthesis ; genetics ; immunology

OBJECTIVETo explore the effect of hypoxia on the fibrosis of corpus cavernosum smooth muscle cells (CCSMC) in SD rats.

METHODSCCSMCs were cultured in vitro, identified by immunohistochemistry, and then exposed to hypoxia at the concentration of 1% O2 for 12, 24, 48 and 72 hours. Those exposed to normal oxygen concentration for the corresponding lengths of time were used as the control. The relative expressions of TGF-131, type I collagen and type DI collagen were determined by RT-PCR.

RESULTSThe in vitro cultured CCSMCs grew well, and the anti-a-smooth muscle actin monoclonal antibodies were positive on immunohistochemical staining. The relative expression levels of TGF-beta1, type I collagen and type mI collagen were positively correlated with the time of hypoxia interference within 48 hours, and did not increase further with prolonged exposure.

CONCLUSIONWhen exposed to hypoxia, the relative expressions of TGF-beta1, type I collagen and type mI collagen in the CCSMCs of SD rats increased with the length of time, and reached the peak at 48 hours. Hypoxia can cause fibrosis of CCSMCs in SD rats.

Animals ; Cell Hypoxia ; Cells, Cultured ; Collagen Type I ; metabolism ; Extracellular Matrix ; Fibrosis ; Male ; Muscle, Smooth ; cytology ; pathology ; Oxygen ; metabolism ; Penis ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

von Willebrand factor-cleaving protease (vWF-cp) is a newly identified metalloproteinase. The activity of vWF-cp would vary in different physiological or pathological states. To explore activity detectron of von Willebrand factor-cleaving protease and its clinical application, the vWF-cp activity was measured by a sensitive enzyme-linked immunoadsorbent assay to detect the residual collagen binding activity (R-CBA) of von Willebrand factor before and after digestion with vWF-cp. Moreover, its activity deficiency in patients with thrombotic thrombocytopenic purpura (TTP) and solid tumors was also investigated. The results showed that the residual collagen binding assay was sensitive enough to measure the serum or plasma vWF-cp activity in 87 health individuals, 79 patients suffering from TTP and solid tumors. The coefficient of variation within and between the batches was were 3.60% and 8.35%, respectively. The serum and plasma vWF-cp activity in health individuals was (79.47 +/- 10.78)% (n = 53) and (78.79 +/- 9.17)% (n = 30), respectively, whereas the vWF-cp activity in patients with TTP, benign and malignant tumors was significantly decreased (P values were less than 0.001, 0.03 and 0.001, respectively). It is concluded that the vWF-cp activity in plasma or serum of patients with TTP and solid tumors markedly decrease, especially in patients with TTP. Assay of the vWF-cp activity using R-CBA is a simple method.
ADAM Proteins ; blood ; metabolism ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; enzymology ; Stomach Neoplasms ; diagnosis ; enzymology ; von Willebrand Factor ; metabolism

OBJECTIVETo investigate the potential association between factor VIII inhibitor development and polymorphisms of tumor necrosis factor-α (TNF-α)-308 and cytotoxic T-lymphocyte associated protein-4 gene in Chinese Han patients with hemophilia A (HA).

METHODSThe single base change polymorphism in TNF-α and CTLA-4 gene was analyzed in 140 Chinese Han patients with hemophilia A who have been treated with plasma-derived FVIII concentrates and 108 normal controls by using PCR-restrictive fragment length polymorphism (RFLP). All of the HA patients' plasma samples were measured by modified-Nijmegen assay simultaneously.

RESULTSIn HA patients, G/G genotype, G/A genotype and A/A genotype were detected in 118 (84.3%), 18 (12.8%) and 4 cases (2.9%) respectively; C/C genotype, C/T genotype and T/T genotype were detected in 108 (77.2%), 30 (21.4%) and 2 cases (1.4%) respectively. The difference in the genotype frequencies between HA patients and controls was nonsignificant (P > 0.05). Patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 7.519, 95% CI = 3.168 - 17.844). Severe HA patients who were carriers of homozygotes for A allele had a higher risk of inhibitor development compared with those who were not (OR = 8.163, 95% CI = 2.521 - 26.434). There was no statistical difference in the risk of inhibitor development between the patients who were carriers or not (OR = 1.586, 95% CI = 0.729 - 3.450).

CONCLUSIONTNF-α-308 gene polymorphism is significantly associated with inhibitor development in Chinese Han patients with severe hemophilia A. TNF-α gene may be a useful marker and potential modulator of the immune response to replacement therapy for hemophilia A patients.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; CTLA-4 Antigen ; genetics ; Child ; Child, Preschool ; Genotype ; Hemophilia A ; genetics ; Humans ; Infant ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Tumor Necrosis Factor-alpha ; genetics ; Young Adult

Hemophilia B (HB) is a recessive X-linked inherited disorder, the pathogenesis of HB is deficiency or functional abnormalities of coagulation factor IX, which is caused by F9 gene mutations. To explore the mechanism of its molecular pathology, 40 patients with HB were studied with polymerase chain reaction (PCR) and direct sequencing. The diagnosis of HB patients were based on clinical manifestation and deficient factor IX activity in plasma. DNA was routinely extracted from peripheral blood cells of the patients and their relatives, all the 8 exons and their flanking boundaries were amplified by PCR, and the PCR products were screened by direct sequencing. Mutations which were found in study need to exclude polymorphism. The results showed that 34 mutations were confirmed in 40 HB patients, including 6 nonsense mutations, 24 missense mutations, 2 splice site mutations and 2 frame mutations for 1 or 2 nucleotide insertion. After retrieved, 4 missense mutations and 1 frameshift mutation were found for the first time. Among the 34 mutations, 2 mutations in signal peptide, 7 mutations in propeptide and gla domain, 7 mutations in epidermal growth factor-like domain, 3 mutations in activation domain, 15 mutations in serine protease or catalytic domain. It is concluded that gene analysis can directly explain molecular mechanism of hemophilia B and also provides the foundation for further studies to the function of coagulation factor IX. There is obvious heterogeneity in F9 gene mutation and missense mutation is still the main way of mutation, which are closely related to clinical features. DNA sequencing and linkage analysis are efficient methods for HB carriers and prenatal gene diagnosis.
Base Sequence ; DNA Mutational Analysis ; Factor IX ; genetics ; Hemophilia B ; diagnosis ; genetics ; Humans ; Male