Biomedical subjects
Publications and source records attributed to A Prado.
B16 mouse melanoma cells in monolayers may be satisfactorily fused with 50% PEG 1500. However, pre-treatment with detergents in solution at low concentrations significantly increases PEG fusion, up to 8-fold in some instances, without impairing cell viability. The practical and mechanistical implications of this finding are discussed.
The solubilizing effects of the non-ionic detergent Triton X-100 have been examined on three membranous systems, namely rabbit sarcoplasmic reticulum, Halobacterium purple membrane and gramicidin A-phosphatidylcholine liposomes. The loss of membrane structure has been assessed through changes in suspension turbidity, while chemical analysis has revealed the differential solubilization of proteins and lipids. Solubilization data obtained on the above three systems are compared with previously published values concerning other membrane preparations. Also, solubilization of sarcoplasmic reticulum by Triton X-100 is monitored by Fourier-transform infrared spectroscopy and, similarly, purple membrane-surfactant interaction is studied using visible spectroscopy. The biochemical and spectroscopic data may be rationalized assuming a three-stage model of membrane-detergent interaction, incorporation of surfactant monomers into the membrane; disruption of the bilayer into mixed micelles, and separation of lipid and protein.
Membranes from a thermophilic eubacterium, Thermus sp. strain SPS 11, isolated from thermal springs of São Pedro do Sul spa (Portugal), are characterized for having two main polar lipids, a glycolipid (GL) with four monosaccharide residues, which at 73 degrees C accounts for 95% of the carbohydrate in the total lipid extracts, and a glycophospholipid (PL) which at 73 degrees C accounts for about 90% of the lipid phosphorous. A complex mixture of carotenoids (CA) makes up 11% by weight of the total membrane lipids. The branched fatty acyl chains (iso C15 and iso C17) comprise about 90% of the alifatic moieties of the polar lipids of this bacterium. Moreover, when the growth temperature increases from 50 to 73 degrees C there is an increase of the iso C17/ iso C15 ratio and of the GL/PL ratio. We have studied the biophysical properties of bilayers (as multilamellar liposomes) prepared from GL, PL and the mixtures of PL, GL and CA in proportions found in the membranes of bacteria growing at their optimal growth temperature, using polarization of DPH fluorescence, low and wide-angle X-ray diffraction and differential scanning calorimetry. The three techniques agree in showing the presence of a broad phase transition from a gel (L beta) phase to a liquid-crystal (L alpha) phase between 8 and 30 degrees C, for all the lipid dispersions studied except for the GL. Although all the dispersions studied form a bilayer structure at all the temperatures studied, only the mixture of the three components (PL, GL + CA) avoids the phase separation present in the mixtures of PL + CA at temperatures lower than 30 degrees C and PL + GL at temperatures lower than 55 degrees C. Our results are compared with those of Pinheiro et al. (1978) obtained with the 31p-NMR technique and applied to the study of the same samples.
A simple approach to correct the constricted ear is described using a V-Y advancement to lengthen the periphery. Then through a posterior incision, an anterior exposure allows the creation of cartilaginous folds with scoring and sutures under direct vision.
The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in São Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.
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Nevus comedonicus is and uncommon abnormality. Clinical and histopathologic characteristics are described. Also the histogenesis is discussed. Eleven new cases are reported. A tretinoin solution or cream produced excellent improvement.
The lipid and fatty acid composition of a strain of Streptomyces antibioticus has been studied as a function of culture age and glycine concentration in the growth medium. Under all conditions, the main polar lipids were phosphatidylethanolamine, cardiolipin and phosphomannoinositides in order of decreasing abundance; no ornithinolipids were detected. Acylglucoses and menaquinones were found among the nonpolar lipids. The main fatty acids present were anteiso 15:0 and anteiso 17:0. The lipid composition of the cells varied with the age of the culture, but no uniform pattern of variation was found in the cultures grown on different amounts of glycine. Among the cells harvested at the end of the exponential phase of growth, those grown on 2% glycine give the highest yield of protoplast formation. These cells were found to contain low amounts of nonpolar lipids and of phosphatidylethanolamine, and high proportions of anteiso fatty acids. We propose that the membrane bilayer of these cells, because of its peculiar lipid composition, is particularly stable and fluid. As a consequence, the yield and stability of derived protoplasts should be increased.
In order to improve the cytological criteria for the diagnosis of cutaneous T-cell lymphomas (CTCL), a number of morphometric assessments in semithin sections have been performed on the dermal infiltrates of twenty-one cases of overt CTCL (group I) and twenty-two cases of well-defined benign dermatoses (group II). In each biopsy, an average of 250 cells were measured for perimeter (P), surface area (S), 'nuclear shape index' (NSI), mean surface and mean NSI. We also determined the percentage of typical 'highly cerebriform lymphocytes' (HCL). Cells of group I patients were found to have mean S and mean NSI that differed significantly from those of group II. HCL were found to have a mean NSI value of less than 0.40. The diagnostic value of these measurements was confirmed in the group I patients. Nineteen of them had a mean NSI value of less than 0.61, together with a mean S greater than 14 sq. micron. None of the twenty-two patients of group II had such values. The discriminating power of these criteria was then tested retrospectively on another group of initially controversial patients who presented with suspected CTCL (group III patients). Nine have since evolved into overt CTCL (group III M) while the other ten have remained benign (group III B). All group III B patients and five patients of group III M were correctly assigned using the above morphometric criteria. This method could improve the diagnosis of the early stage of cutaneous T-cell lymphoma.
The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 mumol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.
Coupled and uncoupled respiration, and energy-dependent phosphate swelling have been studied in rat liver mitochondria in the presence of various concentration of Triton X-100. Detergent concentrations up to 10(-5) M do not affect any of the processes under study. At 10(-5) M, Triton X-100 produces a slight decrease of coupled respiration and a considerable inhibition of mersalyl-induced shrinking in swollen mitochondria. Increasing the surfactant concentration to 10(-4), coupled as well as uncoupled O2 consumption is decreased, succinate-dependent phosphate swelling is inhibited and an energy-dependent phosphate swelling in the absence of valinomycin is observed. At 2 X 10(-4) M. Triton X-100, ATP- dependent phosphate swelling is abolished, and passive swelling may be induced by various ions. Higher detergent concentrations do not allow observation of any of these events. On the basis of these results, a model of membrane-detergent interaction is proposed.
The carbohydrate histochemistry of normal human kidney has been investigated by the use of four peroxidase-labeled lectins at the light and electron microscopic level. The results show that the lectin of Lotus tetragonolobus, specific for l-fucose, binds exclusively to the proximal convoluted tubules of the nephron. While peanut and soybean lectins, specific for D-galactose and N-acetyl-D-galactosamine, respectively, are confirmed to the collecting ducts, wheat germ lectin, specific for sialic acid and N-acetyl-D-glucosamine, stains several parenchymal structures, including the glomerular capillary wall, particularly its podocyte cell coat. Sialidase digestion reveals strong binding sites for peanut and soybean lectin in the glomeruli. At the ultrastructural level most of the binding is shown to be on the podocyte surface and within the lamina rara externa of the basement membrane. The technique represents a potentially very useful tool for the study of various pathological states in the kidney.
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A histochemical study was performed on light- and electron-microscopic level in a case of Fabry's disease. The patient underwent kidney transplantation for renal failure and died of heart failure 6 months later. Patient's tissues were studied at the light- and electron-microscopic levels with various embedding and staining techniques for lipids and carbohydrates. Two peroxidase-labeled lectins (from Ricinus communis and from Bandeiraea simplicifolia) known to have affinity for alpha- and beta-D-galactose, were strongly reactive with the storage material on frozen sections. The ultrahistochemical and extraction tests showed that the typical granules had a variable reactivity and morphologic characteristics in different cells, probably reflecting different composition. A small number of typical deposits were also observed in the transplanted kidney. This is the first reported case of recurrence of the storage disease in the allograft. Of interest was also the fact that the patient's blood inhibited normal alpha-galactosidase activity, suggesting a possible inhibitor-related mechanism in the pathogenesis of the recurrence.
The lipid and fatty acid composition of Bufo calamytes and Alytes obstetricans livers has been studied. Data for both species are similar, and resemble closely those published for Rana sculenta. Total lipids constitute 20-25% of the total liver dry weight; about one fifth of these are phospholipids; cholesterol makes up 1.7-2.2% of the total liver dry weight. The most abundant phospholipids are phosphatidylcholine and phosphatidylethanolamine. The various lipid cases differ in their fatty acid composition: neutral lipids contain high proportions of palmitoleic and linoleic acids; phosphatidylcholine is characterized by its contents in palmitic acid, whereas cardiolipin contains polyunsaturated fatty acids. Thermal acclimation of toads for 96 h produces but few changes in liver lipid composition.
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The rat brain has been studied between 6 hours after birth and 100 days, using histochemical techniques for acid phosphatase and peroxidase, and silver impregnation for microglial cells. The results indicate that microglia come initially from acid phosphatase-positive cells of the meninges. These invade the nervous parenchyma and transform into ramified microglia. At 3 days of age similar cells are present on the outer surface of the large blood vessels, from which site they migrate into the nervous parenchyma. In 100 days old rats the acid phosphatase-positive cells are practically all pericytes. None of the microglial cells or their precursors give a positive reaction for peroxidase.
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