Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall.

S-protein/vitronectin is a multifunctional glycoprotein interacting with both complement activation and coagulation pathways. Its presence was investigated in 5 femoral and 5 iliac atherosclerotic human arteries, obtained at surgery, by immunoelectron microscopy using an affinity purified rabbit IgG specific for human S-protein/vitronectin. The immunoelectron dense specific deposits were found in both intimal thickenings and fibrous plaques in association with elastic fibers, collagen bundles and cell debris in the vicinity of elastin. Cell debris embedded in the collagen matrix were S-protein/vitronectin negative. S-protein/vitronectin was also absent on intact cells, lipid droplets and cholesterol clefts. All cell debris, however, was positive for C5b-9 deposits suggesting that complement activation had occurred at these sites with or without S-protein/vitronectin interaction. S-protein/vitronectin may play a role in the arterial wall defence by restricting the extent of complement activation.

Plasma factor XIII and platelet factor XIII in hyperlipaemia.

Plasma factor XIII activity measured by a quantitative assay was found to be significantly higher in hypertriglyceridaemic patients (type IV and combined hyperlipoproteinaemia), as compared to normolipaemic controls. No such elevation in plasma factor XIII activity was found in patients with type Ha hyperlipaemia. Plasma pseudocholinesterase was found to parallel the elevated factor XIII activity in hypertriglyceridaemic subjects. In contrast, platelet factor XIII activity was not raised in hyperlipaemic subjects, and plasma factor XIII was found to be normal in a normolipaemic subjects with thrombocythaemia. It was concluded that there is no significant contribution from platelets to plasma factor XIII activity, and that the observed increase in plasma factor XIII in hypertriglyceridaemia results from enhanced hepatic synthesis of the enzyme.

Cells carrying C5b-9 complement complexes in human atherosclerotic wall.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

Clinical studies on plasma fibronectin and factor XIII; with special reference to hyperlipoproteinemia.

When compared to age-matched normal weight normolipidemic control subjects, plasma factor XIII, plasma fibronectin and serum cholinesterase levels were found to be markedly decreased in patients with decompensated cirrhosis of the liver, not significantly changed in hyperlipoproteinemia type IIa (heterozygous subjects) and increased in hypertriglyceridemic subjects (type IIb and IV) as well as in hyperlipidemic nephrotic patients. A possible accelerated hepatic synthesis of certain plasma proteins including factor XIII and fibronectin in patients with the nephrotic syndrome as well as in endogenous hypertriglyceridemia is envisaged. It is also considered that mural thrombi, richer in factor XIII and fibronectin, would be more resistant to fibrinolysis and more readily attached to subendothelial structures.

Comparison of liposome entrapment parameters by optical and atomic absorption spectrophotometry.

Methods for the complete characterization of liposomes prepared by ether-injection are described in detail. The validity of atomic absorption spectrophotometry for measuring markers of trapped volume was checked by comparative determinations of markers with established optical spectrophotometrical methods. The favorable results using atomic absorption spectrophotometry to quantitate the marker Mn2+ are of particular relevance as manganese ion is also the paramagnetic probe in n.m.r. measurements of water permeability of liposomes; our results indicate that in such measurements no other marker need be incorporated.

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.

Ultrastructural aspects of human liver tumours collected by thin needle aspiration biopsy.

Fourteen liver tumor samples, obtained by echographically-guided thin needle aspiration biopsy were electron microscopically studied, pointing out the ultrastructural aspects that allowed the diagnosis of primary and secondary liver neoplasia, and those providing indications for malignancy degrees. Criteria of electronoptic differential diagnosis between dysplastic and malignant lesions are presented. The paper suggests that electron microscopy may be helpful in establishing the forms of liver carcinoma, difficult to be determined only by cytologic examination, and for the early diagnosis mandatory to increase the resectability rate of malignant hepatoma.

Fractionation of human liver mitochondria: enzymic and morphological characterization of the inner and outer membranes as compared to rat liver mitochondria.

The fractionation of human liver mitochondria into inner membrane, outer membrane and matrix material is reported. Compared with rat, human liver mitochondria are more fragile. Fractionation can be achieved in only 2 steps, a digitonin treatment for removal of the outer membrane and centrifugation of the inner membrane plus matrix particles through a linear sucrose gradient resulting in purified inner membranes and matrix.

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from sheep and cow.

1. The diffusional water permeability (p) of sheep and cow red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS). 2. There were no significant differences in the water permeability of sheep and cow RBCs, the values of p being around 3 x 10(-3) cm/sec at 20 degrees C and 5 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies of the effects of PCMBS on water diffusion indicated that in both species the maximal inhibition is reached in 60-90 min at 37 degrees C with 1 mM PCMBS. 4. The degree of inhibition increased as the temperature of measurement decreased, regardless of PCMBS concentration and incubation time. 5. The values of maximal inhibition ranged from 60-70% at 20 degrees C to 50-60% at 37 degrees C in the case of sheep RBCs, and from 45-55% at 20 degrees C to 40-50% at 37 degrees C in the case of cow RBCs. 6. The basal permeability to water of sheep RBCs was estimated as 1.0 x 10(-3) cm/sec at 20 degrees C and 2.2 x 10(-3) cm/sec at 37 degrees C, and that of cow RBCs as 1.6 x 10(-3) cm/sec at 20 degrees C and 2.7 x 10(-3) cm/sec at 37 degrees C. 7. In both species the activation energy of water diffusion was around 23 kJ/mol in control cells and reached values of around 30 kJ/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V--Rabbit (Oryctolagus cuniculus).

1. The diffusional water permeability (Pd) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS). 2. The values of Pd were around 6.3 x 10(-3) cm/sec at 15 degrees C, 7.0 x 10(-3) cm/sec at 20 degrees C, 8.0 x 10(-3) cm/sec at 25 degrees C, 9.1 x 10(-3) cm/sec at 30 degrees C and 10.7 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37 degrees C with 0.5 mM PCMBS. 4. The values of maximal inhibition were around 71-74% at all temperatures. 5. The basal permeability to water was estimated as 1.6 x 10(-3) cm/sec at 15 degrees C, 2.0 x 10(-3) cm/sec at 20 degrees C, 2.4 x 10(-3) cm/sec at 25 degrees C, 2.6 x 10(-3) cm/sec at 30 degrees C, and 3.1 x 10(-3) cm/sec at 37 degrees C. 6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion. 7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart. 8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative nuclear magnetic resonance studies on water diffusional permeability of red blood cells from mice and rats.

1. The diffusional water permeability (P) of mouse and rat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following exposure to various concentrations of p-chloromercuribenzene sulfonate (PCMBS). 2. There were no significant differences in the water permeability of mouse and rat RBCs, the values of P being around 6 x 10(-3)/sec at 20 degrees C and 11 x 10(-3) cm/sec at 37 degrees C. 3. Systematic studies of the effects of PCMBS on water diffusion indicated that the maximal inhibition is reached in 60 min at 20 degrees C with 1 mM PCMBS for the mouse RBCs and with 2 mM PCMBS for the rat RBCs. 4. The values of maximal inhibition ranged from 55-57% at 37 degrees C and reached values around 70% at 10 degrees C. 5. The degree of inhibition increased as the temperature of measurement decreased, regardless of PCMBS concentration and incubation time. 6. The basal permeability to water of mouse RBCs was estimated as 1.8 x 10(-3) cm/sec at 20 degrees C and 4.6 x 10(-3) cm/sec at 37 degrees C, and that of rat RBCs as 2.2 x 10(-3) cm/sec at 20 degrees C and 4.2 x 10(-3) cm/sec at 37 degrees C. 7. In both species the activation energy was around 27 kJ/mol and reached values over 40 kJ/mol after incubation with PCMBS in the conditions of maximal inhibition of water diffusion.