(1/4361) Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.
Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified kappaB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-kappaB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-kappaB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression. (+info)
(2/4361) The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.
According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles. (+info)
(3/4361) R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.
Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay. (+info)
(4/4361) Tyrosine kinase inhibitors and immunosuppressants perturb the myo-inositol but not the betaine cotransporter in isotonic and hypertonic MDCK cells.
BACKGROUND: The sodium/myo-inositol cotransporter (SMIT) and the betaine cotransporter (BGT1) are essential for the accumulation of myo-inositol and betaine, and hence cell survival in a hypertonic environment. The underlying molecular mechanism involves an increase in transcription of the SMIT and BGT1 genes through binding of a trans-acting factor to enhancer elements in the 5' flanking region of both genes, resulting in increased mRNA abundance and increased activity of the cotransporters. Current evidence regarding transcriptional and post-transcriptional regulation indicates that both cotransporters are regulated in parallel. METHODS: To investigate the signal transduction of hypertonic stress, we examined the effect of tyrosine kinase inhibitors and immunosuppressants on the hypertonicity-induced activity of the two cotransporters in Madin-Darby canine kidney (MDCK) cells. RESULTS: None of the agents studied affected BGT1 activity in isotonic or hypertonic conditions. Treatment of MDCK cells with genistein, a tyrosine kinase inhibitor, increased SMIT activity in hypertonic but not isotonic conditions. The stimulation of SMIT by genistein was accompanied by a parallel increase in mRNA abundance. In contrast, treating cells with tyrphostin A23, another tyrosine kinase inhibitor, or cyclosporine A, an immunosuppressant, inhibited SMIT activity in hypertonic cells. FK506, another immunosuppressant, increased SMIT activity, but only in isotonic conditions. CONCLUSIONS: These results provide the first evidence of divergent regulatory pathways modulating SMIT and BGT activity. (+info)
(5/4361) Long-term effects of cyclosporine A in Alport's syndrome.
BACKGROUND: In 1991, our initial results of cyclosporine A (CsA) administration in eight patients with Alport's syndrome were published. A significant decrease in or disappearance of proteinuria and apparently good tolerance to CsA were observed in all patients. METHODS: CsA administration has been maintained in these eight patients with the aim of obtaining further information about the clinical course of the disease. The ages of these eight patients currently range from 15 to 27 years, and the mean duration of treatment is from 7 to 10 years (x = 8.4 years). RESULTS: Renal function has remained stable, with no evaluable changes in serum creatinine levels compared with pre-CsA treatment values. Proteinuria in all patients has either remained negative or are values far lower than pretreatment levels. A second renal biopsy was performed in all patients after five years of CsA administration. No aggravation of the lesion present at the first biopsy or lesions typical of cyclosporine intoxication was observed. CONCLUSIONS: After a mean duration of 8.4 years and with no deterioration in renal function, we found possible beneficial effects of the continued treatment of CsA in patients with Alport's syndrome who present evidence of progression to renal insufficiency. (+info)
(6/4361) Flow-mediated vasodilation and distensibility of the brachial artery in renal allograft recipients.
BACKGROUND: Alterations of large artery function and structure are frequently observed in renal allograft recipients. However, endothelial function has not yet been assessed in this population. METHODS: Flow-mediated vasodilation is a useful index of endothelial function. We measured the diameter and distensibility of the brachial artery at rest using high-resolution ultrasound and Doppler frequency analysis of vessel wall movements in the M mode. Thereafter, changes in brachial artery diameter were measured during reactive hyperemia (after 4 min of forearm occlusion) in 16 cyclosporine-treated renal allograft recipients and 16 normal controls of similar age and sex ratio. Nitroglycerin-mediated vasodilation was measured to assess endothelium-independent vasodilation. Brachial artery blood pressure was measured using an automatic sphygmomanometer, and brachial artery flow was estimated using pulsed Doppler. RESULTS: Distensibility was reduced in renal allograft recipients (5.31 +/- 0. 74 vs. 9.10 +/- 0.94 x 10-3/kPa, P = 0.003, mean +/- sem), while the brachial artery diameter at rest was higher (4.13 +/- 0.14 vs. 3.25 +/- 0.14 mm, P < 0.001). Flow-mediated vasodilation was significantly reduced in renal allograft recipients (0.13 +/- 0.08 vs. 0.60 +/- 0.08 mm or 3 +/- 2 vs. 19 +/- 3%, both P < 0.001). However, nitroglycerin-mediated vasodilation was similar in renal allograft recipients and controls (0.76 +/- 0.10 vs. 0.77 +/- 0.09 mm, NS, or 19 +/- 3 vs. 22 +/- 2%, NS). There were no significant differences in brachial artery flow at rest and during reactive hyperemia between both groups. The impairments of flow-mediated vasodilation and distensibility in renal allograft recipients remained significant after correction for serum cholesterol, creatinine, parathyroid hormone concentrations, end-diastolic diameter, as well as blood pressure levels, and were also present in eight renal allograft recipients not treated with cyclosporine. Flow-mediated vasodilation was not related to distensibility in either group. CONCLUSIONS: The results show impaired endothelial function and reduced brachial artery distensibility in renal allograft recipients. The impairments of flow-mediated vasodilation and distensibility are not attributable to a diminished brachial artery vasodilator capacity, because endothelium-independent vasodilation was preserved in renal allograft recipients. (+info)
(7/4361) Nephrotic syndrome as a clinical manifestation of graft-versus-host disease (GVHD) in a marrow transplant recipient after cyclosporine withdrawal.
GVHD is one of the most frequent complications of BMT and recently nephrotic syndrome (NS) has been described as a manifestation of chronic GVHD. Here, we present an AA patient who developed NS 1 year after BMT when cyclosporine was stopped. Renal biopsy showed focal sclerosis associated with membranous deposits. He also had other clinical manifestations of chronic GVHD: sicca-like syndrome and colestasis. After 15 days of CsA therapy, he experienced a remarkable improvement in the NS and GVHD as a whole. We comment on immunological mechanisms that could be involved in the pathogenesis of this manifestation. (+info)
(8/4361) Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: how specific is specific?
BACKGROUND: Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed. METHODS: In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA assay and compared with the Emit assay with the old and new pretreatments, the TDx monoclonal and polyclonal assays, the AxSYM, and HPLC. All samples were from patients with liver and/or kidney transplants. RESULTS: The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9-12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac RIA paralleled the Emit results. In contrast to the mean differences, substantial (>200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between -7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function). CONCLUSION: Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern. (+info)