Stem cell factor/c-kit signaling promotes the survival, migration, and capillary tube formation of human umbilical vein endothelial cells

c-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (相似文献   

Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses

The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.     

Transgenic mice expressing osteopontin in hepatocytes as a model of autoimmune hepatitis

Osteopontin, a crucial factor for Th1 immune response, is expressed in stellate cells and macrophages activated in injured liver. To clarify the role of osteopontin in inflammatory changes in the liver, we attempted to establish transgenic mice expressing osteopontin in hepatocytes. Mouse osteopontin cDNA, cloned from concanavalin-A-stimulated spleen cells in C57BL/6 mice, was constructed into the vector containing serum amyloid-P component promoter. This construction was microinjected into fertilized eggs of C57BL/6 mice, and 4 lines of the transgenic mice were obtained. Western blotting and immunohistochemistry revealed that osteopontin was expressed in hepatocytes, but not in non-parenchymal cells, in the transgenic mice. The mean osteopontin concentrations in the liver and plasma in the mice were 13 and 2.6 times higher than those in negative littermates. Antinuclear antibody was positive in the plasma in 50% of the transgenic mice. In the transgenic mice later than 12 weeks of age, mononuclear cell infiltration in the liver developed, and these cells were positive for CD8 and HLA-DR. Plasma ALT activity was increased with focal necrosis in hepatic lobules in the transgenic mice later than 24 weeks of age. The transgenic mice expressing osteopontin in hepatocytes may be useful as a model of autoimmune hepatitis.     

Genetic polymorphims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients

BACKGROUND AND AIMS: Osteopontin, an extracellular matrix protein with RGD motif, is shown to be a cytokine essential for Th1 immune response initiation. Genetic polymorphisms in the osteopontin gene (OPN) determine the magnitude of immunity against rickettsial infection in mice. Similar polymorphisms, if present also in human beings, might affect hepatitis activity in those infected with HCV. METHODS: Blood was collected from 176 patients with chronic hepatitis C. SNPs in the promoter region of OPN were analyzed in 20 patients by direct sequencing of DNA fragments amplified by PCR and in 156 patients by Invader assay. Ninety-five patients compatible to evaluation criteria were classified into three groups depending on maximal serum ALT levels during the observation periods at least for 2 years as follows; lower than 30IU/L (low-activity group), between 30 and 80IU/L with no hepatoprotective treatment (medium-activity group), and higher than 80IU/L irrespective of hepatoprotective treatment (high-activity group). RESULTS: There were 16, 19, and 60 patients in the low-, medium-, and high-activity groups, respectively. Four SNPs (nt -155, -443, -616, and -1748) were detected in the promoter region of OPN. Among them, the SNP at nt -443 (C or T) was a novel one and showed an association with hepatitis activity in our patients: T/T homozygosity was found in 2 (13%), 8 (42%), and 25 (44%), and C/T heterozygosity in 12 (75%), 8 (42%), and 23 (40%), in the low-, medium-, and high-activity groups, respectively. The other 3 SNPs already known showed linkage disequilibrium with D(') and r(2) greater than 0.937 to each other without correlation to disease activity. CONCLUSIONS. OPN promoter region SNP at nt -433 may be a useful marker reflecting hepatitis activity in chronic hepatitis C patients.     

Real-time relationships in intraluteal release among Prostaglandin F2alpha, endothelin-1, and angiotensin II during spontaneous luteolysis in the cow

It is well known that prostaglandin F(2alpha) (PGF(2alpha)) is a physiological luteolysine, and that its pulsatile release from the endometrium is a luteolytic signal in many species. There is now clear evidence that the vasoactive peptides endothelin-1 (ET-1) and angiotensin II (Ang II) interact with PGF(2alpha) in the luteolytic cascade during PGF(2alpha)-induced luteolysis in the cow. Thus, we investigated the local secretion of PGF(2alpha), ET-1, and Ang II in the corpus luteum (CL) and their real-time relationships during spontaneous luteolysis in the cow. For this purpose, an in vivo microdialysis system (MDS) implanted in the CL was utilized to observe local secretion changes within the CL microenvironment. Each CL of cyclic Holstein cows (n = 6) was surgically implanted with MDS capillary membranes (18 lines/6 cows) on Day 15 (estrus = Day 0) of the estrous cycle. The concentrations of PGF(2alpha), ET-1, Ang II, and progesterone (P) in the MDS samples were determined by enzyme immunoassays. The intraluteal PGF(2alpha) secretion slightly increased from 12 h after the onset of luteolysis (0 h) and drastically increased (by about 300%) from 24 h. Intraluteal ET-1 secretion increased from 12 h. Intraluteal Ang II secretion was elevated from 0 h and was maintained at high levels (about 180%) toward estrus. In each MDS lines (in the same microenvironment) within the regressing CL, the local releasing profiles of PGF(2alpha), ET-1, and Ang II CL positively correlated with each other (P < 0.05) at high proportions in 18 MDS lines (PGF(2alpha) vs. ET-1, 44.4%; PGF(2alpha) vs. Ang II, 55.6%; ET-1 vs. Ang II, 38.9%). In contrast, there was no clear relationship among these substances released into different MDS lines implanted in the same CL (with different microenvironments). In conclusion, we propose that the increase of PGF(2alpha), ET-1, and Ang II within the CL during luteolysis is a common phenomenon for both PGF(2alpha)-induced and spontaneous luteolysis. Moreover, this study illustrated the in vivo relationships in intraluteal release among PGF(2alpha), ET-1, and Ang II during spontaneous luteolysis in the cow. The data suggest that these vasoactive substances may interact with each other in a local positive feedback manner to activate their secretion in the regressing CL, thus accelerating and completing luteolysis.     

Analysis of times to repeated events in two-arm randomized trials with noncompliance and dependent censoring

This article develops randomization-based methods for times to repeated events in two-arm randomized trials with noncompliance and dependent censoring. Structural accelerated failure time models are assumed to capture causal effects on repeated event times and dependent censoring time, but the dependence structure among repeated event times and dependent censoring time is unspecified. Artificial censoring techniques to accommodate nonrandom noncompliance and dependent censoring are proposed. Estimation of the acceleration parameters are based on rank-based estimating functions. A simulation study is conducted to evaluate the performance of the developed methods. An illustration of the methods using data from an acute myeloid leukemia trial is provided.     

Higher viscosity participates in the regulation of coronary flow via nitric oxide and indomethacin-sensitive contracting factor

Few studies have reported on the association of viscosity with coronary circulation. We evaluated the change in coronary flow after dextran was added to a perfusion solution to increase viscosity in isolated rat hearts. We also measured NOx- production induced by the change in shear stress in the coronary effluent, as a marker of NO synthesis. The baseline coronary flow was not influenced by the presence of either the cyclooxygenase inhibitor indomethacin, the thromboxane A2 (TXA2)-prostaglandin H2 (PGH2) receptor antagonist ONO-3708, or the TXA2 synthase inhibitor OKY-046. After exposure to solution containing 0.5% dextran, the coronary flow first decreased and then gradually increased until 10 min. The initial decrease in coronary flow was inhibited by indomethacin, ONO-3708, and OKY-046 individually. The gradual increase was completely inhibited by the NO inhibitor L-NAME, but not by indomethacin or ONO-3708. OKY-046 partially inhibited the increase. NOx- levels in the effluent were higher after the dextran solution was administered, and the increased NOx- levels were inhibited by L-NAME. The increased NOx- levels were not inhibited by inhibitors of the cyclooxygenase pathway. It appears that a higher viscosity of perfusion solution induced a gradual increase in NO production and was associated with increased production of indomethacin-sensitive contracting factor.     

Glycosylation site for chondroitin sulfate on the neural part-time proteoglycan, neuroglycan C

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate (CS) proteoglycan that is expressed predominantly in the central nervous system (CNS). NGC dramatically changed its structure from a proteoglycan to a nonproteoglycan form with cerebellar development, whereas a small portion of NGC molecules existed in a nonproteoglycan form in the other areas of the mature CNS, suggesting that the CS glycosylation of NGC is developmentally regulated in the whole CNS. As primary cultured neurons and astrocytes from cerebral cortices expressed NGC in a proteoglycan form and in a nonproteoglycan form, respectively, CS glycosylation seems to be regulated differently depending on cell type. To investigate the glycosylation process, cell lines expressing a proteoglycan form of NGC would be favorable experimental models. When a mouse NGC cDNA was transfected into COS 1, PC12D, and Neuro 2a cells, only Neuro 2a cells, a mouse neuroblastoma cell line, expressed NGC bearing CS chains. In PC12D cells, although three intrinsic CS proteoglycans were detected, exogenously expressed NGC did not bear any short CS chains just like NGC in the mature cerebellum. This suggests that the addition of CS chains to the NGC core protein is regulated in a manner different from that of other CS proteoglycans. As the first step in investigating the CS glycosylation mechanism using Neuro 2a cells, we determined the CS attachment site as Ser-123 on the NGC core protein by site-directed mutagenesis. The CS glycosylation was not necessary for intracellular trafficking of NGC to the cell surface at least in Neuro 2a cells.     

In situ photoaffinity labeling of the target protein for lembehyne A,a neuronal differentiation inducer

A C(36) linear acetylene alcohol, lembehyne A (LB-A), induces neuronal differentiation against neuroblastoma cells morphologically and also functionally. The differentiation and cytostatic effect induced by LB-A was specific to neuroblastoma, Neuro 2A cells. To identify the target protein for LB-A, a radioactive photoaffinity probe, [(125)I]18-(2'-azido-5'-iodo-benzoyloxy)-LB-18 ([(125)I]azido-LB-18), was synthesized. As a result of in situ labeling experiments against Neuro 2A cells, a protein of M(r) 30 kDa was photolabeled specifically. This labeling was inhibited in the presence of LB-A or the active analogs of LB-A, whereas the inactive analogs showed no inhibitory effect on this labeling. These results suggest that this protein of M(r) 30 kDa is the target protein for LB-A and may play an important role for the neuronal differentiation in neuroblastoma, Neuro 2A cells.