Biomedical subjects

E F Soto

Publications and source records attributed to E F Soto.

At least 37 records · Page 2Linked to original sources

PubMed1998

Sustained neonatal hyperthyroidism in the rat affects myelination in the central nervous system.

We have carried out a study of the effects of sustained neonatal hyperthyroidism on myelin and on the oligodendroglial cells, in an effort to obtain further insight into the molecular mechanisms underlying the action of thyroid hormones on the central nervous system (CNS). Expression of the mRNAs of myelin basic protein (MBP) myelin proteolipid protein (PLP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), transferrin, and c-Jun was investigated in 10- and 17-day-old normal and hyperthyroid rats, using Northern blot analysis. At 10 days of age, the levels of all the explored mRNAs were markedly higher in the experimental animals. The mRNA of transferrin showed a ninefold increase over control values, suggesting the possibility that this putative trophic factor might act as one of the mediators in the action of thyroid hormones. At 17 days of age on the other hand, the levels of all the mRNAs decreased markedly, reaching values below control, except for c-Jun, which remained higher than in normals. At 70 days of age, hyperthyroid rats showed clear evidence of myelin deficit, in agreement with previous results of our laboratories (Pasquini et al.: J Neurochem 57: Suppl S124, 1991). Immunocytochemistry of 70-day-old rat brain tissue sections showed a substantial reduction in the amount of MBP-reacting structures and a marked decrease in the number of oligodendroglial cells. Although the above-mentioned results could be the consequence, as proposed by Barres et al. (Development 120:1097-1108, 1994) and Baas et al. (Glia 19:324-332, 1997) of a premature arrest in oligodendroglial cell proliferation followed by early differentiation, the persistent high levels of expression of c-Jun, together with the dramatic decrease in the number of oligodendrocytes, suggested the possibility that prolonged hyperthyroidism could activate apoptotic mechanisms in the myelin forming cells. Using propidium iodide-labeled isolated oligodendroglial cells, we found, by flow cytometry, a significant increase in the number of apoptotic/hypo-diploid propidium iodide-positive cells. These results indicate that one of the actions of sustained levels of thyroid hormones in the neonate rat is to increase oligodendroglial cell death by apoptosis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1997

Single intracranial injection of apotransferrin in young rats increases the expression of specific myelin protein mRNA.

Transferrin (Tf) is a possible regulator of oligodendrocyte development in vitro (Espinosa de los Monteros et al., 1989). At least two different mechanisms may account for the effects of Tf on myelin synthesis. It may act as a trophic factor and enhance the formation of new myelin sheaths. Tf may also induce the synthesis of myelin proteins in the central nervous system. We recently demonstrated that a single intracranial injection of apotransferrin (aTf) in young rats induces an increased myelination (Escobar Cabrera et al., 1994). In the present study, we investigated the in vivo effect of aTf on the expression of mRNAs of specific myelin genes. Three-day-old rats were injected intracranially with aTf and killed at different ages after injection. Total brain RNA was isolated, and the expression of different mRNAs was analyzed by Northern blot. The amount of mRNAs of myelin basic protein and of 2'-3' cyclic nucleotide 3'-phosphohydrolase were markedly increased in the experimental animals, whereas myelin proteolipid protein mRNA did not show differences relative to controls. These results indicate that in the animals treated with aTf, there is a differential effect on the expression of certain specific myelin protein genes. They also suggest that aTf might exert its action at the posttranscriptional level and/or by direct transcriptional regulation of the genes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1995

Oxidative damage to proteins and lipids of CNS myelin produced by in vitro generated reactive oxygen species.

Purified myelin isolated from 70-day-old rats was submitted to nonenzymatic peroxidative systems containing 100 microM FeCl3.6H2O, 100 microM ascorbic acid, and 100 microM CuSO4.6H2O 10 mM H2O2 in order to investigate the extent of damage produced by reactive oxygen species (ROS). Iron and copper catalyzing systems were selected because of the known importance of these metals in producing free radical chain reactions in biological membranes (Halliwell and Gutteridge: "Free Radicals in Biology and Medicine," Oxford: Clarendon Press, 1989). Our findings show that: (1) although after 1 hour of peroxidation, an important level of thiobarbituric acid-reactive substances (TBARS) was detected, polyunsaturated fatty acids (20:2; 20:4; 22:4 and 22:6) were markedly affected only after 14 hours of incubation; (2) protein thiol groups were very sensitive to the attack of ROS generated by copper but resistant to iron-generated ROS; (3) aggregation of myelin proteins produced by peroxidation could be prevented by sulfhydryl (SH)-reducing agents, and (4) as a consequence of these modifications, compact myelin suffered disruption of its intraperiodic line. In conclusion, our results demonstrate that this unique membrane of the central nervous system (CNS) is highly vulnerable to oxidative stress and that this susceptibility to oxidative damage could be prevented, at least partially, by the use of SH-protective molecules.

Animals↗

PubMed1995

Increased susceptibility to degradation by trypsin and subtilisin of in vitro peroxidized myelin proteins.

We examined the possibility that the peroxidative damage to central nervous system myelin produced by reactive oxygen species (ROS), could modify the susceptibility of its proteins to the proteolytic action of proteases such as trypsin and subtilisin. Purified myelin membranes obtained from adult rat brains were "in vitro" peroxidized by two non-enzymatic systems: Fe3+ plus ascorbic acid and Cu2+ plus hydrogen peroxide. Myelin proteins were severely affected by peroxidation. There was an increase in the amount of carbonyl groups (CO), accompanied by an enhanced susceptibility to degradation by trypsin and subtilisin of myelin basic proteins (MBP) and of the major proteolipid protein (PLP). The effect upon the degradation of myelin protein is a possible consequence of the appearance in the structure of myelin proteins of peroxidative modifications that contribute to the recognition by proteolytic enzymes. This hypothesis is supported by the fact that if peroxidation of myelin membranes is done in the presence of EDTA, both CO formation and increased sensitivity to enzymatic breakdown disappear. These results suggest that the appearance of abnormal post-translational modifications in the myelin membrane produced by peroxidation could constitute a putative mechanism of modulating the capacity of myelin proteins to be metabolized by proteases.

Animals↗

PubMed1994

Ubiquitin-protein conjugates in different structures of the central nervous system of the rat.

The capacity to form ubiquitin (Ub)-protein conjugates was investigated in the cytosol of different structures of the rat central nervous system (CNS) in order to confirm the presence of this extralysosomal, adenosine triphosphate (ATP)-dependent, protein degradation system as well as its structural localization. Using 125I-Ub, we found that in the presence of ATP, the cytosol obtained from whole brains was able to form high molecular weight Ub-protein conjugates. These conjugates could be detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. The formation of these conjugates was much higher in the cerebral cortex than in the brain stem, which is mainly constituted by white matter, being intermediate in the cytosol isolated from whole brain total homogenates. These results suggested to us that under normal conditions the capacity to form Ub-protein conjugates was mainly located in structures containing neuronal cell bodies. Strong support for this contention was obtained when the cytosol isolated from rat optic nerves or from oligodendroglial cells isolated from whole brain was found to be totally unable to form Ub-protein conjugates. The inability of certain CNS structures to form conjugates with Ub could be attributed, among other reasons, to the lack of enzymes catalyzing the various steps of the Ub degradation system, to the absence of short half-life (target) proteins in those structures, or to the lack of activity of the enzymes catalyzing the reaction due to regulatory control mechanisms operating under normal conditions.

Animals↗

PubMed1994

Single intracerebral injection of apotransferrin in young rats induces increased myelination.

Three-day-old rats were injected intracranially with 210 or 350 ng of apotransferrin (aTf) to study the possible neurotrophic effects of this iron transport protein. Treated animals and appropriate controls were injected with saline, denatured aTf or ovalbumin. Myelin was isolated from the brains and used to study its chemical composition and the protein electrophoretic pattern. Total myelin galactolipids and especially total phospholipids were significantly increased with reference to controls at both ages studied. A slight increase in total cholesterol was also observed. Total myelin proteins were markedly increased both at 10 and 17 days in comparison to controls. Chloroform:methanol-soluble proteins (proteolipids) were only slightly increased. The electrophoretic profile showed that the two main bands corresponding to myelin basic protein (MBP) were relatively increased in the treated animals. The enzymatic activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) was significantly increased. The changes observed were dose- and age-dependent. The injection of aTf was effective only within a short developmental period since animals treated at 20 days of age showed no apparent changes in myelin composition. The actions of aTf injection persisted at least up to 60 days, since at this age the myelin obtained from injected animals still contained higher amounts of total proteins phospholipids and galactolipids in comparison to control animals. The action of aTf appears to be specific for myelin, since no significant effects were observed in membranes of a total brain homogenate. The effects are specific for the apoprotein injected since other proteins (ovalbumin) or heat-denatured aTf were ineffective.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane.

The possibility that cerebroside sulfates and myelin proteolipid (PLP) could be simultaneously located in transport vesicles destined to be assembled in myelin was investigated in the brain of 20 day old rats. The brain was homogenized and fractionated according to Burkart et al. (J Biol Chem 257:3151-3156, 1982) to obtain a microsomal fraction that was further subfractionated in a linear sucrose density gradient following the procedure of Siegrist et al. (J Neurochem 33:497-504, 1979) to obtain a vesicular fraction which has been shown to transport cerebroside sulfates (Burkart et al., as above). This fraction was associated with acid hydrolase activity and had a lipid composition different from that of myelin and microsomal fractions. Studied by slab gel electrophoresis, dot blot, and Western blot analysis, using a highly specific anti-PLP antibody, it was found to contain myelin PLP. In view of previous findings of several laboratories including our own, the presence of myelin proteolipid in a vesicular fraction which is related to the transport of cerebroside sulfates gives further support to the hypothesis that the delivery of both constituents to the myelin membrane could be associated.

Animals↗

PubMed1992

Rat brain fatty acid-binding protein during development.

Cytosolic fatty acid-binding proteins (FABPs) have been described in rat and bovine whole brain. In the present study we investigated the distribution of FABP among white matter and gray matter as well as its changes during development. Fatty acid binding activity was similar in white and gray matter up to 40 days of age. In white matter it showed an age dependent increase thereafter, while in gray matter it remained constant throughout. Gel filtration (Sephadex G-75) of white matter cytosol of adult female rats resolved the fatty acid-binding activity in two peaks: A (Vo) and B (12-14 KDa; FABP). The specific binding activity in the FABP fraction was 10.4 pmol/micrograms of protein. The activity in peak A showed an age-dependent increase which paralleled myelin deposition. In contrast, the activity in the FABP fraction (peak B) remained undetectable up to 40 days of age, increasing thereafter. The differential distribution of cellular brain proteins with the capacity to bind fatty acids in gray matter and white matter suggests that this activity could be related to glial cells or to cell related structures such as myelin.

Aging↗

PubMed1992

Phospholipid composition of oligodendroglial cells during normal development and in 18 day old hyperthyroid and malnourished rats.

The phospholipid composition of isolated oligodendroglial cell perikarya was studied in normal rats during development and in 18 day old malnourished and hyperthyroid rats. Phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipid constituents of oligodendroglial cells. Phospholipid content increased during development, mainly due to an increase of the above mentioned phospholipids. The major changes were observed in sphingomyelin, phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine between 18 and 30 days of age. The phospholipid and protein content per cell was significantly decreased in the oligodendroglial cells isolated from malnourished rats as compared to controls. When data were expressed as a function of total proteins, the composition was similar to that of normal animals. In the hyperthyroid rats on the other hand, there were no changes in the amount of phospholipids per cell, while phospholipids per milligram of total oligodendroglial cell protein were markedly decreased. The changes in myelin composition produced by hyperthyroidism that we have previously described, do not follow closely those produced by this experimental condition in oligodendroglial cells, suggesting that the metabolism of myelin might be to a certain extent, independent of that in the parent cell.

Aging↗

PubMed1990

Neonatal hyperthyroidism in the rat produces an increase in the activity of microperoxisomal marker enzymes coincident with biochemical signs of accelerated myelination.

The effect of neonatal hyperthyroidism produced by injection of tri-iodothyronine (T3) on myelination and on the microperoxisomal population of the brain was studied in young rats. Data on the lipid composition of myelin show that myelinogenesis starts earlier in treated animals. In coincidence with the early appearance of myelin, there is an increase in the population of brain microperoxisomes, indicated by the increase in the activity of two enzymes that have been shown to be located in these organelles: catalase and acyl CoA-dihydroxyacetone phosphate acyl transferase. Double-label experiments using (1,2,3-3H) and (2-3H) glycerol to study the synthesis of glycerophospholipids through the dihydroxyacetone phosphate (DHAP) pathway give further support to the above-mentioned findings and suggest that there is an active participation of microperoxisomes in the synthesis of myelin lipids during the period of myelin formation.

Acyltransferases↗

PubMed1990

Effect of neonatal undernutrition upon cerebroside sulfate degradation in the developing rat brain.

In order to find out if the decreased accumulation of cerebroside sulfates observed in 21-d-old undernourished rats was in part the result of an increased rate of catabolism of these galactolipids, the in vivo degradation of brain cerebroside sulfates was studied in 18-d-old normal and undernourished rats. Two hours after the intracranial injection of the precursor (0 time), the animals were injected intraperitoneally with unlabeled sodium sulfate. Labeled cerebroside sulfates were measured in the brain up to 48 h after the chase. In normal animals, the radioactivity decreased at 24 h and 48 h to 55% and 41%, respectively, of the value obtained at 0 time. In undernourished animals, degradation was negligible, since the radioactivity attained at 0 time remained almost constant up to 48 h. The lack of in vivo degradation of cerebroside sulfates observed in the starved rats cannot be explained by a deficiency of Arylsulfatase A, since the pattern of activity of the enzyme was similar in both groups of animals.

Aging↗

PubMed1989

Inhibition of the synthesis of glycosphingolipids affects the translocation of proteolipid protein to the myelin membrane.

Brain slices obtained from young rats were incubated with different radioactive precursors, in the presence and absence of L-cycloserine (an inhibitor of the synthesis of sphingosine) in order to explore the possibility that transport of proteolipids--and specifically of the major myelin proteolipid PLP--to the myelin membrane could be coupled to the transport of cerebrosides or sulfatides. At a concentration of 0.15 mM L-cycloserine, the incorporation of [3H] glycine into total proteins, proteolipid apoproteins (APL), PLP, and myelin basic proteins (MBP) of the total homogenate was unaffected by the presence of the inhibitor, whereas the incorporation of [3H] serine into glycosphingolipids decreased markedly. Under similar incubation conditions, the entry of labeled APL and of PLP into the myelin membranes in the presence of L-cycloserine decreased markedly (50%) in comparison to controls. Entry of MBP was not affected by the inhibitor. These results indicate that when synthesis of glycosphingolipids is inhibited by L-cycloserine, thus decreasing the availability of cerebrosides and sulfatides, the translocation of PLP to myelin is disrupted, suggesting that its transport through the oligodendroglial cell could be coupled to the transport of glycosphingolipids and, most probably, of sulfatides.

Animals↗

PubMed1987

Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.

Animals↗

PubMed1985

Deacylation of myelin proteolipid protein in organic solvents.

A procedure has been developed for the deacylation of the hydrophobic, myelin proteolipid apoprotein using hydroxylamine in an alkaline organic solvent medium. Complete removal of covalently bound fatty acids was obtained after 4 hr of treatment. After deacylation, no changes could be detected in the electrophoretic pattern or in the number of free sulfhydryl groups. The deacylated apoprotein remains soluble in chloroform-methanol mixtures and is suitable for further physicochemical characterization.

Acylation↗

PubMed1985

Neonatal malnutrition in the rat affects the delivery of sulfatides from microsomes and their entry into myelin.

Brain slices from 18 day old normal and malnourished rats were incubated in the presence of [35S]sulfate to explore its incorporation into sulfatides of a total brain homogenate and the appearance of labeled sulfatides in different subcellular fractions. While the incorporation of label into sulfatides of the total homogenate was similar in both groups of animals, in subcellular fractions separated on a linear sucrose density gradient, labeling of sulfatides in malnourished animals was relatively higher in the region corresponding to the microsomal fraction. Time course incorporation and pulse-chase experiments were carried out to explore the kinetics of labeling of microsomal and myelin sulfatides. In pulse-chase experiments, normal controls showed a decrease in the specific radioactivity of sulfatides in the microsomal fraction after the chase, which was not observed in malnourished animals, while the appearance of labeled sulfatides in the myelin fraction of the latter group of animals was found to be lower than in normals. These results suggest that in neonatal malnutrition there is a defect in the transport of de novo synthesized sulfatides towards myelin or/and a problem in the assembly of these lipids into the myelin membrane.

Animals↗

PubMed1985

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: effect of malnutrition.

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [3H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18-21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain a normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.

Age Factors↗

PubMed1985

Electrophoretic analysis of myelin proteolipid protein and its deacylated form.

Myelin proteolipid protein is known to contain covalently bound fatty acid. To determine the contribution of the fatty acid to the multiple bands observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretic parameters of the proteolipid protein were compared with those of the deacylated form. The relative mobility and proportion of each band, as well as the retardation coefficient and free electrophoretic mobility, were not altered by removal of the fatty acid moiety. Furthermore, the acylated and deacylated forms bound the same amounts of sodium dodecyl sulfate. These data demonstrate that the presence of covalently bound fatty acids does not account for the electrophoretic heterogeneity of the proteolipid.

Acylation↗

PubMed1984

Mechanisms of transport and assembly of myelin proteins.

The present study was carried out in order to obtain further information regarding the mechanism of transport and assembly of myelin proteins in different subcellular fractions isolated from brain slices incubated in vitro with radioactive amino acids under different experimental conditions. It was found that proteolipid protein (PLP) showed a lag in the entry into the myelin membrane, while basic and Wolfgram proteins appeared to be inserted in this structure immediately after their synthesis. Addition of 500 microM colchicine to the incubation medium blocked the transport of PLP, while the entry of the other proteins was not affected. Pulse-chase experiments using cycloheximide suggest that a precursor-product relationship between microsomes, fraction SN4 and myelin exists only for PLP. The results obtained allow us to draw the following conclusions: The delay in the entry of PLP into myelin membrane is probably due to the time required for its transport towards the final site of assembly; the microtubular network of the oligodendroglial cell is directly involved in the transport of PLP; basic and probably Wolfgram proteins follow a route which clearly differs from that of PLP; delivery of myelin proteins from the site of synthesis towards their site of deposition depends, at least, on two different mechanisms of intracellular transport.

Animals↗