Showing papers by "Facultad de Ciencias Médicas published in 1995"

Protein-Energy Malnutrition during Gestation and Lactation in Rats Affects Growth Rate, Brain Development and Essential Fatty Acid Metabolism

Abstract: The influence of feeding a low protein diet to rat dams during gestation and lactation on lipid metabolism in pups was studied. Wistar rats were fed 5, 10, 15 and 25% dietary protein during gestation and lactation. Pup growth was monitored until weaning, and brain weight, protein concentration, proteolipid concentration and total lipid phosphorus concentration of brain were analyzed. The levels of fatty acids in dam milk as well as in pup liver phospholipids and brain prosphatidylcholine and phosphatidylethanolamine were determined. The progressive deprivation of maternal dietary protein produced a reduction in the total saturated fatty acid concentration of dam milk and an increment in the concentration of nonmetabolized linoleic acid. Pup body and brain weights as well as proteolipid, protein and total lipid phosphorus concentrations in brain were reduced in proportion to the degree of dietary protein deficiency. The products:precursor ratio of (n-6) fatty acids in liver phospholipids revealed an impairment in the elongation-desaturation pathway due to maternal protein deficiency. Both (n-6) and (n-3) polyunsaturated fatty acids within brain phosphatidylethanolamine were decreased by reduced maternal dietary protein intake, whereas only the linoleic acid-derived products were similarly affected in the corresponding phosphatidylcholine fraction. These results demonstrate the widespread and profound deleterious effects of low protein levels of maternal diet on the growth rate, brain development and fatty acid metabolism in rat pups.

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice.

Abstract: The nonobese diabetic (NOD) mouse is an animal model that shares a number of clinical, genetic, and immunologic characteristics with human insulin-dependent diabetes mellitus. Since little is known about the morphometric cell profiles in the endocrine pancreas of these NOD animals, it was of interest to assess their changes in morphometry within the pancreatic islet cell types during two stages of this syndrome. Prediabetic (6-week-old) and diabetic (16-week-old) NOD female mice, as well as normal C57BL/6 female mice (15 weeks old), were used. Light microscopic immunocytochemical and morphometric methods were employed to study the endocrine cell populations. The immunoperoxidase technique for the identification of insulin, glucagon, somatostatin, and pancreatic polypeptide, as well as the point-counting method, was used on serial sections of pancreas tissue. Compared to those of normal and prediabetic mice, pancreata from diabetic animals showed a decrease in both the number of islets and the volume density of the endocrine component. Analysis of islet tissue revealed a significant diminution of B-cell volume density, as well as an increased A-, D-, and PP-cell volume density. A parallel variation in the number of B and non-B cells was also found. In addition, when the total pancreatic tissue surface was taken as reference, the fractional area occupied by all the different types of islet cells was seen to be diminished in a variable fashion. We conclude that the diabetic syndrome of NOD mice not only severely affects the B-cell mass, but also causes marked changes in the non-B endocrine-cell populations.

Expression of monoclonal-antibody-defined antigens in fractions isolated from human breast carcinomas and patients' serum.

Abstract: The aim of this study was to examine tissue from patients with breast carcinoma or benign breast disease for the presence of monoclonal-antibody-defined antigens, including the MUC1 mucin and carcinoembryonic antigen CEA. The tests were performed by sodium dodecyl sulphate/polyacrylamide gel electrophoretic separation of proteins, electrophoretic transfer to nitrocellulose membranes and immunostaining with the monoclonal antibodies. Some of the antigens identified are known to circulate at high levels in some but not necessarily all, breast carcinoma patients. Serum from a panel of ten breast cancer patients was subjected to a fractionation procedure designed to release antigen from immune complexes, and again these samples were analysed for the presence of monoclonal-antibody-defined antigens. A high frequency of positive reactions was detected by the anti-MUC1 monoclonal antibody C595 with both breast carcinoma subcellular membrane fractions as well as antigen fractions eluted from circulating immune complexes. No reactions were observed with equivalent materials from benign breast disease samples. The findings illustrate the variability in antigen expression between breast tumours. The data also indicate that a proportion of patients respond to their tumour by the production of antibodies that recognise the MUC1 antigen in their circulation.

Regulatory effect of various steroid hormones on the incorporation and metabolism of [14C]stearate in rat hepatoma cells in culture

Abstract: We have examined the incorporation and metabolism of [14C] stearic acid within the total lipids of HTC rat-hepatoma cells in suspension culture in presence and in absence of steroidal hormone stimulation. Both, glucocorticoids (dexamethasone, cortisol and corticosterone) and mineralocorticoids (deoxycorticosterone and aldosterone) as well as the estrogen β-estradiol and the androgen testosterone enhanced the extent of Δ9 desaturation to oleic acid of the saturated precursors, whereas only the two mineralocorticoids affected the incorporation rate of the exogenous acid into total cellular lipids, thus promoting a little stimulation. Furthermore, all the hormones tested increased the radiolabelling of the total cellular phospholipids except deoxycorticosterone and testosterone, the former having no effect and the latter exerting a moderate inhibition. On the other hand, the incorporation of14C into neutral lipids was stimulated by testosterone, in contrast to the inhibition of this parameter observed exclusively with either the mineralocorticoids or the estrogen. Within the phospholipid subclasses, the radiolabelling of phosphatidylcholine was augmented by means of all the steroids tested save deoxycorticosterone and testosterone, whereas phosphatidylethanolamine exhibited a decrease only in the presence of testosterone. In a similar fashion, within the neutral lipids, the predominating triglyceride fraction was preferentially labelled—at the expense of other subclasses of lesser abundance—upon treatment with the steroids except aldosterone, which exerted no effect. The results obtained were correlated with those changes observed in the mass distribution of the different lipid subclasses either with or without prior hormonal stimulation.

Fatty acid uptake and metabolism in Hep G2 human-hepatoma cells.

Abstract: The aim of this work was to study the fatty acid metabolism of the human-hepatoma cell line Hep G2. The cultured cells were incubated with either a saturated (palmitic, stearic) or a polyunsaturated (linoleic, alpha-linolenic, eicosatrienoic n-6) radioactive fatty acid. The fatty acids were incorporated into all the basic lipid classes as well as into the main phospholipid subclasses in the cellular membranes. All the fatty acids tested provided a source of carbon for lower members of the saturated fatty-acid family or for cholesterol through beta-oxidation and a new cycle of de novo synthesis. Moreover, all radioactive fatty-acid precursors, whether saturated or unsaturated, were anabolized to higher derivatives within their own family. In the case of saturated fatty acids, palmitic and stearic, they were readily monodesaturated to their corresponding products, thus demonstrating the presence of a delta 9 desaturase. Linoleate and alpha-linolenate were both desaturated and elongated to all the subsequent members of their respective n-6 and n-3 families. These latter observations provide evidence for the incidence of desaturation at the 6 and 5 positions along with the existence of an elongating capacity for fatty acids of all families and chain lengths. In addition, the cellular steady-state fatty-acid profile was seen to be significantly different from the spectrum of exogenous fatty acids available in the growth medium. We conclude that the Hep G2 human-hepatoma line represents an appropriate and relevant experimental model system for investigating the fatty-acid metabolism of adult human liver in vivo.

Comparative study of the effect of sodium fluoride and sodium monofluorophosphate on glucose homeostasis in the rat.

Abstract: The effect of acute and chronic administration of sodium fluoride (NaF) or sodium monofluorophosphate (MFP) on the glucose homeostasis of the rat are compared. The oral administration of a single dose of 40 mumol/100 g b.w. of either compound produced similar increases in plasma glucose (up to 1.8 g/l) and diffusible fluoride (up to 130 mumol/l). In long-term experiments (three months of duration), treatment with NaF (a 5 mmol/l solution as the water supply) produced, in the first month of experiment, abnormal glucose tolerance tests and increased plasma diffusible fluoride levels (range: 2-12 mumol/l). Treatment with MFP, on the other hand, did not affect glucose homeostasis; plasma diffusible fluoride was always below 2 mumol/l. The results of these experiments indicate that glucose homeostasis is affected when plasma diffusible fluoride exceeds 5 mumol/l. The basal and glucose-stimulated insulin secretion of isolated Langerhans rat islets (incubated with solutions containing 2, 5, 10 and 20 mumol/l NaF) was significantly inhibited by 5 to 20 mumol/l fluoride. No effect was observed under similar conditions with MFP at concentrations of 2, 5, 10, 20 and 50 mumol/l.

Fatty acid delta 5 desaturation in rat liver cell nuclei.

Abstract: Activity of one of the key enzymes involved in arachidonic acid (20:4 n−6) biosynthesis, the Δ5 desaturase, was found in rat liver cell nuclei. Up to now, it has been shown that the fatty acid desaturases are located exclusively in the endoplasmic reticulum. Similarly to what happens with microsomal enzyme the nuclear Δ5 desaturase enzyme was only fully active in the presence of a cytosolic factor. In this condition it reached a specific activity of 50 pmol 20:4 n−6 formed/min/mg of protein. This fact would imply that purified nuclei like purified microsomes lack a soluble cytosol factor necessary for the total desaturation reaction expression. Besides the nuclear Δ5 desaturase has an optimal pH of 7.6 and is inhibited by 1 or 10 mM KCN. Low long chain acyl-CoA synthetase activity that catalyzes the formation of 20:3 n−6-CoA, was also found in liver nuclei. This step would be essential in nuclear desaturation since when ATP and/or CoA (necessary for the acylation reaction) are omitted from the incubation mixture, the desaturation reaction does not take place.

Occupational rhinoconjunctivitis and bronchial asthma due to Phoenix canariensis pollen allergy.

Abstract: We report a case of occupational bronchial asthma and rhinoconjunctivitis caused by Phoenix canariensis (PC) pollen. The canary palm is a type of palm tree, belonging to the Arecaceae family, which is widely distributed in frost-free regions as an ornamental tree. Our patient was referred because he suffered symptoms of bronchial asthma, rhinoconjunctivitis, and contact urticaria when pruning dried leaves from PC during the pollination months. The skin prick test (SPT) with a PC pollen extract was positive, as was the specific IgE to PC pollen determined by Phadezym RAST, indicating an IgE-mediated sensitization. The nonspecific bronchial provocation test (BPT) performed with methacholine disclosed a mild bronchial hyperreactivity, and specific BPT with PC pollen elicited an immediate fall of 25% in FEV1 with respect to baseline. On RAST inhibition studies, a significant cross-reactivity was found between PC pollen and date palm (P. dactylifera) pollen. These results suggest that PC pollen could be a potential allergen in PC-growing areas.

Changes induced by non-enzymatic glycosylation of IGF-binding protein-3: effects on its binding properties and on its modulatory effect on IGF-I mitogenic action.

Abstract: The aim of this study was to demonstrate the feasibility of in vitro non-enzymatic glycosylation of IGF-binding protein-3 (IGFBP-3) and whether this process affects its binding properties and its modulatory effect on IGF-I mitogenic activity. Swiss 3T3 fibroblasts were cultured and the IGFBP-3 released into the medium (CM) glycated with either labelled or unlabelled glucose. Parallel glycation studies were performed using standard human IGGBP-3. Both species of IGFBP-3 became effectively glycated in a dose-dependent manner. Glycated IGFBP-3 bound larger amounts of 125 I-labelled IGF-I than its non-glycated form. According to Scatchard analysis this effect might be due to an increase in the number of binding sites of the IGFBP-3 molecule rather than to changes in its affinity constants, which remain unchanged. Preincubation of fibroblasts with CM containing IGFBP-3 for 16 h before the addition of IGF-I enhanced the stimulatory effect of the hormone on thymidine incorporation into cell DNA. This potentiation was blunted when in vitro glycated instead of non-glycated IGFBP-3 was employed. These results provide further evidence of the in vitro glycation of IGFBP-3 and demonstrate that tits process affects both its binding properties and its enhancing effect on IGF-I mitogenic activity. These changes may explain, at least partially, the development of many alterations observed in poorly controlled diabetic patients

Relationship between frame size and fatness in children and adolescents.

Abstract: In order to evaluate variation in body fatness by category of frame size (small, medium, and large) during growth, a sample of 7,286 Cuban children and adolescents (3,721 males, 3,565 females) 5-20 years was surveyed. Fatness was estimated from skinfold thicknesses at four sites: subscapular, triceps, suprailiac, and posterior calf. Elbow breadth was used as an estimate of frame size. Generally, the larger the frame size, the greater the amount of subcutaneous fat regardless of age and sex. The results suggest that in the assessment of obesity and its associated risk, in children and youth, frame size, given by categories of elbow breadth, should be taken into account. © 1995 Wiley-Liss, Inc.

Electron Microscopic Immunolocalization of Caltrin Proteins in Guinea Pig Seminal Vesicles

Abstract: Caltrins, the small, basic proteins of the seminal vesicle secretion that inhibit calcium transport into epididymal spermatozoa, and consequently the onset of the acrosome reaction and the hyperactivated motility, were localized in the epithelial cells and the lumen of the seminal vesicles of the guinea pig by an immunocytochemical procedure and electron microscopy. Rabbit antisera against each protein (caltrin I or II), and goat anti-rabbit IgG antiserum labeled with colloidal gold were used to detect the caltrin immunoreaction. The subcellular distribution of the gold labeling was occasionally localized in the rough endoplasmic reticulum but mainly within big secretory vacuoles containing low electron-dense material, which are components of the Golgi complex known as condensing vacuoles. These are involved in the intracellular transport, storage, and discharge of secretory proteins. Gold-labeled material released to the lumen was also detected. There was no clear evidence that the discharge was mediated by an exocytotic process. Immunoreaction was observed neither in the electron-dense core nor in the electron-lucent halo of the typical secretory granules of the epithelial cells of the seminal vesicles. Using light microscope immunocytochemistry, intense positive immunoreactivity was detected in the material secreted to the lumen but not on the epithelial cell layer. Only those cells undergoing a degenerative process and showing a picnotic nucleus and condensed cytoplasmic matrix exhibited detectable immunoreaction when gold label and silver intensification were applied. The same distribution of the immunoprobes was obtained by electron or light microscopy when antiserum to either I or II was used. It would appear that the two caltrin proteins of the guinea pig are synthesized in the rough endoplasmic reticulum of the epithelial cells and transported quickly to the Golgi complex where the secretory vacuoles (condensing vacuoles) are formed. The proteins are transported by the secretory vacuoles to the apical ends of the cells to be discharged into the lumen.

Studies on very-high-density lipoprotein of Triatoma infestans hemolymph in relation to its function as free fatty acid carrier

Abstract: The function of Triatoma infestans very-high-density lipoprotein (VHDL) as a free fatty acid transport protein was analyzed. Lipophorin (HDLp) and VHDL are the unique hemolymphatic proteins able to transport free fatty acids (FFA). The transfer of this lipid species between HDLp and VHDL was studied using 14 C-palmitic acid-labeled VHDL or 14 C-palmitic acid-labeled HDLp as donor substrate and the same unlabeled lipoproteins as acceptor substrate. The VHDL is more effective as acceptor of 14 C-FFA from HDLp rather than donor of 14 C-FFA to HDLp. When 14 C-palmitic acid-labeled VHDL was incubated with either fat body or testicle, it was observed that the 14 C-palmitic acid was taken up by both tissues and incorporated into their lipid components.

Uptake and Release of Ca2+ in Chemically Skinned Aortic Strips from Spontaneously Hypertensive (SHR) and Normotensive (WKY) Rats.

Abstract: The uptake and release of Ca2+ were studied in EGTA-skinned aortic strips from spontaneously hypertensive rats (SHR strain: SAP=191±5mmHg, n=27) and normotensive control rats (WKY strain: SAP=131±2mmHg, n=25). 45Ca uptake was measured as a function of time (0.5 to 30min.), at pCa 6.6, in the presence of 10mM of K oxalate. Skinned aortic strips of SHRs accumulated more Ca2+ after 30min of uptake than those of WKY rats (0.66±0.05 vs 0.52±0.03nmole.mg-1 wet tissue; p<0.05). A lower activity of the transport system in the hypertensive group was evidenced by the fraction of these maximal uptake values accumulated after 2 minutes of uptake, 56% compared with 98% in the normotensive group. 45Ca release was assayed in skinned aortic strips preloaded for 30 minutes with 45Ca in the absence of K oxalate and desaturated with washing solutions containing 3nM free Ca2+. 30mM of caffeine, 5μM of norepinephrine or 10μM of IP3 resulted in greater increases in the rates of Ca2+ efflux in WKY than in SHR aortic strips. Net effluxes of Ca2+ upon stimulation with all these drugs were statistically significant only in the hypertensive group due to its slightly but consistently higher Ca2+ content. Changes in both rate of efflux and net efflux induced by 30mM of caffeine could be blocked by 0.6mM of ryanodine. The sarcoplasmic reticulum is characterized in the genetically hypertensive rats by a low transport activity of its Ca2+-ATPase, a high Ca2+ content and a Ca2+ release mechanism with low responsiveness to stimulation by caffeine, norepinephrine and IP3

Comparação das frações HDL e LDL colesterol como fatores de risco para a aterosclerose carotídea

Abstract: In order to find out whether there exists a relationship between HDL and LDL serum levels and atherosclerotic plaques in the carotid artery (CA), a prospective study was carried out involving 125 patients. They were aleatorily included, of both sexes and age between 45-75 years old. HDL and LDL serum levels were measured as well CA ultrasonographic mode B investigated. It was verified that the number of patients having atherosclerotic plaques in CA was inversely proportional to the HDL levels. We came up with the same ratio when the HDL levels were compared to the number of patients having CA stenosis. These results were statistically signifcant (x2=6.57 and x2=9.24 respectively; critical x2=5.99 to α =5%). A direct ratio was also found out in the relationship between the presence of atherosclerotic plaques in the CA and the LDL serum levels (greater in patients with plaques). However no relationship between LDL levels and the occurrence or not of CA stenosis in patients having plaques was demonstrated. The results were not statistically significant (x2=0.97 and x2=0.41, respectively, critical x2=5.99 to α =5%). The obtained results seem to be at least in part in agreement with literature findings. Statistically significative results in the comparison of HDL and LDL levels with the grade of stenosis of the CA were not found out (x2=11.78 and x2=4.03, respectively; critical x2=12.59 to α =5%).

Effect of pH upon Ca(2+)-ATPase activity of rat pancreatic islets: its possible contribution to the inhibitory effect of different insulin secretagogues

Abstract: This work was undertaken in an attempt to elucidate the possible mechanism by which insulin secretagogues produce a fast and transient drop in the Ca(2+)-ATPase activity of the pancreatic islet membrane. For this purpose, the enzyme activity was measured in either homogenates or partially purified membranes of islets previously incubated under different experimental conditions. Ca(2+)-ATPase activity measured in homogenates of islets preincubated with 8 mM glucose decreased significantly compared to control islets incubated with 2.8 mM glucose. The inhibition was also observed when the enzyme activity was measured in homogenates of islets preincubated with 2.8 mM glucose plus 20 mM propionic acid as well as with glucose 2.8 mM in a buffer equilibrated with a gas mixture of O2 and either 12% or 30% CO2. Ca(2+)-ATPase activity decreased significantly in partially purified islet membranes preincubated for 3 min with glucose (2 and 8 mM), 15 mM KCl and 2 mM tolbutamide. These substances did not affect the Ca(2+)-ATPase activity when added directly to the enzyme assay medium. The enzyme activity also decreased when measured in membranes preincubated at pH 6.5. The addition of 1 mM ATP to the preincubation medium protected the Ca(2+)-ATPase activity from the inhibition induced by glucose, KCl and tolbutamide as well as from the one produced by acidic pH in the medium. On account of these results, we suggest that insulin secretagogues, as well as either acidification of B-cell cytosol or islet membrane incubation medium, produce changes at the islet membrane level which promote a decrease in the Ca(2+)-ATPase activity. A shift of the E1-E2 equilibrium of the phosphoenzyme towards E1 may account for such decreased activity. Changes in Ca(2+)-ATPase activity could either favour the decrease or the increase in the cytosolic concentration of Ca2+ in B-cells. Therefore, negative and positive modulation of its activity might allow Ca(2+)-ATPase to play a role in the switch-on and -off mechanism for intracellular Ca2+ signal regulation of B-cell secretion of insulin.