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A new method for the cytofluorometric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzim idazolcarbocyanine iodide (JC-1)
No full textA new method for the cytofluorimetric analysis of mitochondrial membrane potential in intact cells has been developed by using the lipophilic cationic probe 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzim idazolcarbocyanine iodide (JC-1), whose monomer emits at 527 nm after excitation at 490 nm. Depending on the membrane potential, JC-1 is able of forming J-aggregates that are associated with a large shift in emission (590 nm). The color of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. In two human cell lines (K562 and U937), we have studied by flow cytometry the changes in membrane potential provoked by the K+ ionophor valinomycin, a drug known to affect mitochondrial membrane potential, while the K+/H+ ionophor nigericin, known to affect intracellular pH but not mitochondrial membrane potential, was used as control. The incubation with valinomycin for 10 min. at 37°C in a low K+ medium provoked a marked and dose-dependent reduction in JC-1 greenish orange fluorescence, while nigericin had no effect. © 1993 Academic Press, Inc
Aortic elastin abnormalities in osteogenesis imperfecta type II.
No full textSkin and aortic samples from two patients who died by lethal perinatal Osteogenesis Imperfecta (O.I.) were studied by optical and electron microscopy and compared with similar samples from two normal human fetuses and one newborn child. No significant abnormalities were observed in the dermis of O.I. patients apart from small differences in the diameter of reticular collagen fibrils. On the contrary, in the aortas of both patients collagen fibrils were significantly smaller than in the controls; moreover, elastin lamellae were deeply altered and consisted of roundish aggregates of elastin, massively permeated by cytochemically recognizable glycosaminoglycans. As identical features were described in experimental lathyrism by using inhibitors of the enzyme lysyl oxidase (Pasquali Ronchetti et al., 1984), the conclusion is reached that in the two cases of lethal perinatal O.I. examined, a severe lysyl oxidase deficiency could account for the observed ultrastructural abnormalities of elastin and that, besides defects of collagen type I, additional alterations of cellular metabolism might be responsible for the clinical heterogeneity of the disease
Elastin fiber-associated glycosaminoglycans in beta-aminopropionitrile-induced lathyrism.
No full textRuthenium red and toluidine blue O precipitates were described associated with lathyritic elastic fibers in aortas of chickens treated with beta-aminopropionitrile fumarate (I. Pasquali-Ronchetti, C. Fornieri, I. Castellani, G. M. Bressan, and D. Volpin (1981). Alterations of the connective tissue components induced by beta-aminopropionitrile. Exp. Mol. Pathol. 35, 42-56). In this report evidence is given that these precipitates reveal the presence of proteoglycans, as they are completely removed by 5 M guanidine-HCl incubation and by specific enzymatic digestions. In particular, proteoglycans associated with the poorly cross-linked lathyritic elastin can be removed by testicular hyaluronidase, chondroitinase ABC, heparitinase, and nitrous acid treatments, whereas they are rather resistant to streptococcal hyaluronidase and chondroitinase AC. On the contrary, proteoglycans of the matrix or associated with collagen fibers are particularly sensitive to these latter enzymatic treatments. The conclusion is reached that glycosaminoglycans associated with beta-aminopropionitrile-induced lathyritic elastin (i) are different from those of the matrix or associated with collagen, and (ii) include mainly dermatan and heparan sulfates
Plasma lipoproteins in rats with experimental biliary obstruction. II. An ultrastructural study.
No full textAcute biliary obstruction in the rat is associated with abnormalities of the ultrastructure of plasma lipoproteins. 1.1. The d < 1.006 g/ml fraction contains two types of particles: (a) vesicles having a mean diameter of 42.0 nm and (b) spheroidal particles which have a mean diameter of 80.0 nm. The latter show surface irregularities which make these particles appear as spherical bodies with holes.2.2. The d 1.019–1.063 g/ml fraction is heterogeneous. Gel filtration on 2% agarose allows the characterization of three components designated subfraction I, II and III respectively. Subfraction I is composed of large aggregates of spherical and vesicular particles. Subfraction II consists of 50.0–80.0 nm vesicles which show strong similarities to artificially prepared emulsions of phospholipids in water. Subfraction III contains particles which have a mean diameter of 20.0 nm and resemble low density lipoproteins of normal rats.3.3. The d 1.063–1.125 g/ml fraction contains 35.0 nm discoidal particles which tend to form ruleaux with a periodicity of 4.9 nm. This fraction also contains a few vesicles and 10.0 nm spherical particles similar to those found in the corresponding fraction of normal rat
Pseudoxanthoma elasticum (PXE): ultrastructural and biochemical study on proteoglycan and proteoglycan-associated material produced by skin fibroblasts in vitro.
No full textPseudoxanthoma elasticum is a genetic disease characterized by progressive mineralization of elastic fibers. Previous studies suggested that other components, apart from elastin, might be involved in the alterations of this connective tissue disorder (Martinez-Hernandez and Huffer, 1974; Pasquali Ronchetti et al., 1981; 1986). Evidence is presented that proteoglycan metabolism is altered in PXE-affected patient. Urinary GAGs suggests an increased degradation of glucosamine-containing GAGs in the patient. Pulse and chase experiments on in vitro skin fibroblasts indicated a decreased rate of synthesis of [35SO4] containing GAGs or an increase of their turnover rate in PXE. Moreover, when PGs produced from skin fibroblasts were identified by ultracentrifugation and gel filtration in associative conditions, PXE fibroblasts produced a significantly higher amount of the high molecular weight fraction of sulfated PGs. This high molecular weight material was present both in the medium and in the matrix and disappeared under dissociative conditions or after treatment with hyaluronidase or with pancreas elastase. By electron microscopy, PXE fibroblasts appeared to produce and secrete an enormous amount of toluidine blue 0 positive material organized as filaments and amorphous masses. These data are in agreement with previous observations of the presence of abnormal masses of microfilaments, in the dermis of PXE patients, which were sensitive to hyaluronidase and partially to trypsin and elastase (Pasquali Ronchetti et al., 1986). The results seem to confirm that at least some of the alterations of connective tissues in PXE are due to abnormal PGs metabolism and to their tendency to form abnormal aggregates in the extracellular space
Proteoglycan alterations in skin fibroblast cultures from patients affected with Pseudoxanthoma elasticum
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