Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Thromb Res ; 126(5): 406-13, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20864147

ABSTRACT

Plactin, a family of cyclopentapeptides of fungal origin, enhances fibrinolytic activity by promoting of single-chain urokinase-type plasminogen activator (scu-PA) activation on the cell surface. For this activity, factor(s) in the blood plasma is absolutely required. In the previous studies, we identified prothrombin as a plasma cofactor involved in this mechanism, while the presence of another independent cofactor was suggested. The objective of this study was to identify the second cofactor and investigate the mechanism involved. Using plactin-affinity and ion-exchange chromatographies, we purified plasma hyaluronan-binding protein (PHBP) ~4,000-fold from human plasma as an independent plactin cofactor. PHBP, at ~10nM, was effective in plactin-dependent promotion of scu-PA activation by U937 cells. PHBP is a serine protease that is produced as a single-chain proenzyme (pro-PHBP) and autoproteolytically converted to an active two-chain form. Pro-PHBP was comparable to PHBP in activity to promote plactin-dependent scu-PA activation by U937 cells. Plactin enhanced both cellular binding and autoproteolytic activation of pro-PHBP. The two activities were obtained with a plactin concentration at ~30µM, which resulted in a significant increase in intrinsic fluorescence and self association of pro-PHBP. Thus, it is suggested that such changes account for both enhanced cellular binding and autoactivation of pro-PHBP, resulting in an enhancement of scu-PA activation.


Subject(s)
Peptides, Cyclic/metabolism , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , Enzyme Precursors/metabolism , Humans , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Binding , Serine Endopeptidases/pharmacology , U937 Cells , Urokinase-Type Plasminogen Activator/metabolism
2.
Biochim Biophys Acta ; 1761(7): 709-16, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16815093

ABSTRACT

Adiponectin is an adipose tissue-specific secretory protein known to be an insulin-sensitizing protein. In this study, we generated adiponectin sense and antisense transgenic (Tg) mice to investigate whether adiponectin plays a role in the regulation of energy homeostasis during the growth stage. Spontaneous motor activity of antisense Tg mice were markedly reduced during fasting, particularly in young female mice, compared with wild type (Wt) and sense Tg mice. Furthermore, both body weight and adipose tissue mass of the antisense female Tg mice drastically reduced during fasting. To examine the relationship between the collapse of abdominal white adipose tissue (WAT) and serum adiponectin level, we measured the expression of genes related to energy expenditure, such as uncoupling protein (UCP). Notably, the mRNA of UCP1 in the WAT of antisense Tg female mice was markedly less than that of Wt mice and the UCP1 mRNA was strongly increased during fasting. These findings suggest that the serum adiponectin is important to maintaining energy homeostasis under energy shortage conditions, such as over female pubertal development.


Subject(s)
Adiponectin/metabolism , Energy Metabolism , Adiponectin/genetics , Adipose Tissue/metabolism , Aging/physiology , Animals , Body Weight , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA, Antisense , Energy Metabolism/genetics , Fasting , Female , Gene Expression Regulation, Developmental , Ion Channels , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mice , Mice, Transgenic , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Organ Specificity , Sex Factors , Uncoupling Protein 1 , Uncoupling Protein 2
3.
Int J Oncol ; 29(1): 209-15, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16773202

ABSTRACT

The kringle domain is a triple loop structure present in angiostatin and endostatin. The disulfide bond-linked kringle architectures have been known to be essential for anti-angiogenic activity. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which consists of three epidermal growth factor (EGF) domains, a kringle domain, and a serine protease domain. PHBP can be cleaved autocatalytically to generate activity and is highly expressed in the human blood and liver. To determine the anti-angiogenic activities of PHBP, we purified recombinant mouse PHBP from stable cell line overexpressing PHBP and used protein in vivo and in vitro angiogenesis assays. We found that recombinant PHBP inhibits not only angiogenesis in vivo in chorioallantoic membrane (CAM) assay but also the basic fibroblast growth factor (bFGF)-induced proliferation, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependant manner. Moreover, we found that the kringle domain of PHBP was essential for the anti-angiogenic action of PHBP by the deletion mutants. These findings unravel a new function of PHBP as an inhibitor of the proangiogenic phenotype of vascular endothelial cells and demonstrate that the kringle domain of PHBP might be a potent novel inhibitor of activated endothelial cells in vitro and in vivo.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Endothelial Cells/drug effects , Neovascularization, Physiologic/drug effects , Serine Endopeptidases/pharmacology , Animals , Cell Movement , Cell Proliferation , Cells, Cultured , Chick Embryo , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/drug effects , Collagen , Dose-Response Relationship, Drug , Drug Combinations , Endothelial Cells/cytology , Fibroblast Growth Factor 2/pharmacology , Humans , Kringles/genetics , Laminin , Mutation , Proteoglycans , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Umbilical Veins/cytology , Umbilical Veins/drug effects
4.
Clin Chem ; 52(6): 1045-53, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16574760

ABSTRACT

BACKGROUND: Several proteolytically derived fragments from the proline-rich region (PRR) of human inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) have been identified by surface-enhanced or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS or MALDI-TOF-MS) as potential disease markers. METHODS: Previously, we developed a SELDI-based immunoassay that can simultaneously distinguish and quantify multiple isoforms/variants of a protein/peptide of interest. In this study, we used this high-throughput approach to quantify and characterize the extensive fragmentation within the PRR of human serum ITIH4 and determined its association with different disease conditions. The ITIH4-related fragments were first immunocaptured by use of beads coupled with peptide-specific antibodies. The eluates were then studied by SELDI-TOF-MS. In addition, freshly collected and immediately processed serum and plasma samples were used to analyze the ex vivo stability of these ITIH4 fragments. RESULTS: Human serum ITIH4 was shown to be extensively proteolytically processed within the PRR, and its fragmentation patterns were closely associated with different disease conditions. Fragmentation patterns were generally consistent with cleavages by endoprotease followed by exoprotease actions. Observed fragments changed little under different assay conditions or blood collection and processing procedures. CONCLUSIONS: The fragmentation patterns within the PRR of human serum ITIH4 are associated with different disease conditions and may hold important diagnostic information. These fragmentation patterns could be useful as potential biomarkers for detection and classification of cancer.


Subject(s)
Glycoproteins/blood , Adult , Aged , Amino Acid Sequence , Biomarkers, Tumor/blood , Blood Proteins/chemistry , Breast Neoplasms/blood , Colonic Neoplasms/blood , Diabetes Mellitus/blood , Female , Glycoproteins/chemistry , Humans , Immunoassay , Male , Middle Aged , Molecular Sequence Data , Ovarian Neoplasms/blood , Pancreatic Neoplasms/blood , Prostatic Neoplasms/blood , Proteinase Inhibitory Proteins, Secretory , Serum , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
5.
FEBS Lett ; 532(3): 283-8, 2002 Dec 18.
Article in English | MEDLINE | ID: mdl-12482580

ABSTRACT

Placentas of mice lacking p57(Kip2) expression have trophoblastic hyperplasia. To elucidate the mechanism underlying this phenomenon, we studied expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). Immunohistochemical analysis with anti-VEGF antibodies indicated that VEGF expression was stronger and more clearly detectable in placentas of p57(Kip2) null embryos compared to wild-type placentas. PlGF showed no significant differences between placentas of p57(Kip2) null and wild-type embryos. In quantitative analysis, placentas of p57(Kip2) null embryos showed higher VEGF messenger (m)RNA and protein levels than did wild-type placentas. PlGF mRNA and protein levels were not significantly different. These findings suggest that VEGF is involved in the hyperplasia that occurs in placentas of p57(Kip2) null embryos.


Subject(s)
Endothelial Growth Factors/biosynthesis , Gene Expression Regulation, Developmental , Intercellular Signaling Peptides and Proteins/biosynthesis , Lymphokines/biosynthesis , Nuclear Proteins/genetics , Placenta/metabolism , Pregnancy Proteins/metabolism , Animals , Cyclin-Dependent Kinase Inhibitor p57 , Hyperplasia , Immunoblotting , Immunohistochemistry , Mice , Mice, Transgenic , Placenta Growth Factor , Protein Isoforms , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
6.
BMC Med Genet ; 3: 6, 2002 Jul 29.
Article in English | MEDLINE | ID: mdl-12147176

ABSTRACT

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method. METHODS: Single-nucleotide polymorphisms (SNPs) of the inter-alpha-trypsin inhibitor family heavy chain-related protein (IHRP) gene were analyzed using a fluorescence-adapted SSCP method. The method was constructed from two procedures: 1) a fluorescent labeling reaction of PCR fragments using fluorescence-adapted primers in a single tube, and 2) electrophoresis on a non-denaturing polyacrylamide gel. RESULTS: This method was more economical and convenient than the single-stranded conformational polymorphism (SSCP) methods previously reported in the detection of the labeled fragments obtained. In this study, eight SNPs of the IHRP gene were detected by the fluorescence-adapted SSCP. One of the SNPs was a new SNP resulting in an amino acid substitution, while the other SNPs have already been reported in the public databases. Six SNPs of the IHRP were associated with two haplotypes. CONCLUSIONS: The fluorescence-adapted SSCP was useful for detecting and genotyping SNPs.

SELECTION OF CITATIONS
SEARCH DETAIL
...