Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite.
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OBJECTIVES: This study examined the effects of endotoxin on cardiac contractility in human myocardium. BACKGROUND: In animal myocardium, endotoxin and cytokine treatment led to enhanced inducible nitric oxide synthase (iNOS) expression and contractile dysfunction. Effects in human myocardium are unknown. METHODS: Left ventricular myocardial preparations from failing (n = 18) and nonfailing (n = 5) human hearts were incubated for 6 and 12 h in tyrode solution or in tyrode plus lipopolysaccharides (LPS), with LPS plus N(G)-mono-methyl-L-arginine (L-NMMA), with LPS plus hemoglobin or with LPS plus the superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). Force of contraction in response to isoprenaline (0.001 to 3 micromol/liter) was determined in electrically stimulated muscle preparations. The iNOS mRNA expression was examined by in situ hybridization and by polymerase chain reaction. The cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay. RESULTS: Isoprenaline concentration dependently increased force of contraction. Six and 12 hours of LPS treatment of failing myocardium decreased maximum inotropic response to isoprenaline by 54% (p = 0.009) and by 69% (p = 0.0023), respectively. In nonfailing myocardium, 12 h of LPS treatment decreased maximum inotropic effect of isoprenaline by 66% (p < 0.001). The LPS effects were attenuated by L-NMMA, hemoglobin and also Tiron. The iNOS mRNA was expressed in all LPS-treated preparations but also in most control myocardial preparations. In situ hybridization revealed iNOS expression within cardiac myocytes. There was no increase in myocardial cGMP content in response to endotoxin. CONCLUSIONS: Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by enhanced iNOS activity and release of nitric oxide (NO). Consecutive reaction of NO with superoxide and formation of peroxynitrite may contribute to the decrease in force of contraction. (+info)
Requirement of type III TGF-beta receptor for endocardial cell transformation in the heart.
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Transforming growth factor-beta (TGF-beta) signaling is mediated by a complex of type I (TBRI) and type II (TBRII) receptors. The type III receptor (TBRIII) lacks a recognizable signaling domain and has no clearly defined role in TGF-beta signaling. Cardiac endothelial cells that undergo epithelial-mesenchymal transformation express TBRIII, and here TBRIII-specific antisera were found to inhibit mesenchyme formation and migration in atrioventricular cushion explants. Misexpression of TBRIII in nontransforming ventricular endothelial cells conferred transformation in response to TGF-beta2. These results support a model where TBRIII localizes transformation in the heart and plays an essential, nonredundant role in TGF-beta signaling. (+info)
Urate synthesis in the blood-sucking insect rhodnius prolixus. Stimulation by hemin is mediated by protein kinase C.
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Hemin is a catalyst of the formation of reactive oxygen species. We proposed that hematophagous insects are exposed to intense oxidative stress because of hemoglobin hydrolysis in their midgut (Petretsky, M. D., Ribeiro, J. M. C., Atella, G. C., Masuda, H., and Oliveira, P. L. (1995) J. Biol. Chem. 270, 10893-10896). We have shown that hemin stimulates urate synthesis in the blood-sucking insect Rhodnius prolixus (Graca-Souza, A. V., Petretsky, J. H., Demasi, M., Bechara, E. J. H., and Oliveira, P. L. (1997) Free Radical Biol. Med. 22, 209-214). Once released by fat body cells, urate accumulates in the hemolymph, where this radical scavenger constitutes an important defense against blood-feeding derived oxidative stress. Incubation of Rhodnius fat bodies with okadaic acid raises the level of urate synthesis, suggesting that urate production can be controlled by protein phosphorylation/dephosphorylation. Urate synthesis is stimulated by dibutyryl cAMP and inhibited by N(2((p-bromocinnamil)amino)ethyl)-5-isoquinolinesulfonamide (H-89), an inhibitor of protein kinase A, as well as activated by the protein kinase C activator phorbol 12-myristate 13-acetate. In the presence of hemin, however, inhibition of urate synthesis by H-89 does not occur, suggesting that the hemin stimulatory effect is not mediated by protein kinase A. Calphostin C completely inhibits the hemin-induced urate production, suggesting that the triggering of urate antioxidant response depends on protein kinase C activation. This conclusion is reinforced by the observation that in fat bodies exposed to hemin, both protein kinase C activity and phosphorylation of specific endogenous polypeptides are significantly increased. (+info)
Ultrastructure and viability of isolated bovine preantral follicles.
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Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes. (+info)
Glucocorticoids and insulin promote plasminogen activator inhibitor 1 production by human adipose tissue.
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Plasminogen activator inhibitor 1 (PAI-1) is likely to play a role in vascular disease, primarily in subjects with android obesity. It has been demonstrated that PAI-1 is overexpressed in adipose tissue from obese subjects and that visceral adipose tissue produced more PAI-1 than subcutaneous fat. In the present study, the effect of insulin and glucocorticoids, which are key mediators of adipose tissue metabolism, was examined in relation to PAI-1 synthesis by human adipose tissue explants (HAT), collagenase isolated human adipocytes (IHA), cultured human stromal cells (cSC), and differentiated adipocytes from the murine clonal cell line 3T3-F442A. A significant increase in PAI-1 antigen release (1.5-fold) from HAT was detectable after 16 h of treatment with insulin concentrations of at least 10(-8) mol/l. This was associated with a PAI-1 mRNA increase. Concomitant addition of insulin (10(-8) mol/l) to forskolin (5 x 10(-5) mol/l) reversed the decrease in PAI-1 antigen caused by forskolin alone. No effect on PAI-1 antigen was observed when insulin was incubated with IHA or cSC. 3T3 F442A cells were sensitive to insulin with a four- and twofold increase in PAI-1 antigen and mRNA levels, respectively, after 16 h of stimulation with 10(-8) mol/l. Dexamethasone (DXM) significantly enhanced PAI-1 antigen and mRNA expression by HAT (1.5- and 2.5-fold increase, respectively) at concentrations of at least 10(-8) mol/l. A higher stimulation was observed with IHA (sevenfold increase) and with the differentiated 3T3 F442 cell line. Cortisol was found to be less potent than DXM. No effect was observed when glucocorticoids were incubated with cSC. Coincubation of HAT with insulin (10(-7) mol/l) and DXM (10(-7) mol/l) led to an additive effect on PAI-1 synthesis. These results support the hypothesis that PAI-1 expression in human adipose tissue is controlled by insulin and glucocorticoids and may help to explain the increase in plasma PAI-1 levels observed in patients with android obesity. (+info)
Regulation of forestomach-specific expression of the murine adenosine deaminase gene.
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The maturation of stratified squamous epithelium of the upper gastrointestinal tract is a highly ordered process of development and differentiation. Information on the molecular basis of this process is, however, limited. Here we report the identification of the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult mouse, Ada is highly expressed in the terminally differentiated epithelial layer of upper gastrointestinal tract tissues. The data reported here represent the identification and detailed analysis of a 1. 1-kilobase (kb) sequence located 3.4-kb upstream of the transcription initiation site of the murine Ada gene, which is sufficient to target cat reporter gene expression to the forestomach in transgenic mice. This 1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level comparable to the endogenous Ada gene. This expression is localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites. (+info)
Study of the cell biology and biochemistry of cherubism.
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AIMS: To establish whether the multinucleate cells in lesions of patients with cherubism are also osteoclasts and if this is the case whether they were responsive to calcitonin; to carry out cytogenetic studies on two members of the same family affected by cherubism in an attempt to identify any major chromosomal defects; and to perform an in-depth modern biochemical study of four children in the same family. SUBJECTS AND METHODS: Four related children with cherubism were studied. Tissue taken from one of the children at elective decompression of an optic nerve was submitted to in vitro bone resorption studies. Cytogenetic studies were done on two of the children and biochemical studies on all four. RESULTS: The multinucleate cells in the cherubic lesions were shown to be osteoclasts since they synthesised tartrate resistant acid phosphatase, expressed the vitronectin receptor, and resorbed bone. Bone resorption by the cultured multinucleate cells was significantly inhibited by calcitonin. High resolution cytogenetic studies failed to detect any chromosomal abnormalities in two children with cherubism. The biochemistry profile of all four children with cherubism showed that serum calcium, parathyroid hormone, parathyroid related hormone, calcitonin, and alkaline phosphatase were within normal levels. Urine analysis of pyridinium and deoxypyridinium cross links, hydroxyproline, and calcium in relation to urine creatinine were measured to assess bone resorption in these children, and the values were at the upper end of the normal range in all four. CONCLUSIONS: Further studies are required to determine whether calcitonin treatment will control this grossly deforming disease until the time when the physiological changes that occur at puberty rectify the pathology. It is not recommended that biochemical markers of bone resorption are used in isolation to monitor the activity of cherubism in individuals because the results are based on a small number of children and because of reports of marked interindividual variation in the levels of these markers, particularly in children. (+info)
Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.
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AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage. (+info)