Biomedical subjects

M Lopes-Cardozo

Publications and source records attributed to M Lopes-Cardozo.

At least 37 records · Page 2Linked to original sources

PubMed1991

Interferon-gamma induced IA antigen expression on cultured neuroglial cells and brain macrophages from rat spinal cord and cerebrum.

The inducibility of major histocompatibility complex class II (Ia) antigens on glial cells of the brain suggests that neuroglia have immunoregulatory functions within the central nervous system (CNS), i.e., recognition and presentation of antigens. The aim of the present study was to investigate rat recombinant-interferon-gamma (r-IFN-gamma) induced Ia antigen expression in rat cerebral cultures containing type-1 astrocytes and macrophages, and in rat spinal cord cultures enriched in type-2 astrocytes or oligodendrocytes. We compared induction of Ia antigen expression in glial cell cultures derived from Lewis rats, which are very susceptible to experimental allergic encephalomyelitis (EAE), with those from Wistar rats, which are but modestly EAE susceptible. After 5 days in culture we found in Wistar rat type-1 astrocyte-enriched cultures that Ia antigens were expressed by 19% of the astrocytes, whereas we found that in Lewis rat type-1 astrocyte cultures a considerably higher number of astrocytes expressed Ia antigens (53%). However, no significant difference were found in Ia antigen expression between type-2 astrocytes derived from Wistar rat spinal cord (49%) and Lewis rat type-2 astrocytes (56%). In contrast, in oligodendrocyte-enriched cell cultures derived from either Lewis or Wistar rats no Ia antigen expression was found. Interestingly, we found in type-1 astrocyte-enriched cerebral cultures a large number (approx. 46% of the cells) of brain macrophages (amoeboid microglia), all expressing Ia antigens after treatment with r-IFN-gamma.

Animals↗

PubMed1990

Galactosylceramide sulfotransferase, arylsulfatase A and cerebroside sulfatase activity in different regions of developing rat brain.

The in vivo metabolism of sulfatides was studied in spinal cord and cerebral cortex of developing rat pups. Developmental changes in the rate of sulfolipid synthesis were measured after the intraperitoneal injection of 35SO4(2-). We also measured the accumulation of sulfatides, as well as the profiles of cerebroside sulfotransferase, cerebroside sulfatase and arylsulfatase A in both brain regions as a function of postnatal development. The accumulation of sulfatides was higher in spinal cord than in cerebral cortex. In addition, sulfatide metabolism was more active in spinal cord. In both brain regions, the developmental pattern of 35SO4(2-) incorporation into sulfolipids was closely correlated to the activities of cerebroside sulfotransferase and of arylsulfatase A. The activity of these enzymes was initially low, increased during the period of active myelination and declined thereafter. However, the activity of cerebroside sulfatase, measured with its physiological substrate, [35S]sulfatide, increased during development and did not decline. An explanation for the difference between the developmental profiles of the arylsulfatase A and cerebroside sulfatase reactions (which are supposed to be catalysed by the same enzyme) is proposed.

Aging↗

PubMed1990

A rapid procedure for the preparation of oligodendrocyte-enriched cultures from rat spinal cord.

Spinal cords and cerebra from 7-day-old rat pups were compared as tissue sources for the isolation of oligodendrocytes and for studies on the development of these cells in culture. After 1 day in culture the serum-containing medium was replaced by a chemically-defined medium, which contained a cocktail of hormones that stimulated oligodendrocyte development. The cultures were characterized with various immunocytochemical markers; monoclonal A2B5 for bipotential glial progenitor cells, anti-galactocerebroside (GC) serum for oligodendrocytes, and anti-glial fibrillary acidic protein (GFAP) serum for astrocytes. The number of positive cells was counted and expressed as a percentage of total cells. At 1 day in culture the cell cultures from spinal cord contained 30% GC+ cells, increasing to 90% after 7 days in culture. In cultures derived from cerebra the percentage of GC+ cells was always lower than in cultures from spinal cord. In cerebral cultures GFAP+ cells increased from 15% at 1 day in culture to 30% at 7 days in culture, whereas it remained low in spinal cord cultures. The activity of oligodendroglial marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase was followed during development in culture. The specific activity increased rapidly in both types of culture but was more than threefold higher in cultures derived from spinal cord. This procedure yields, within one week and without subculture, primary glial cultures from rat spinal cord, that are highly enriched in oligodendrocytes (greater than or equal to 90%; 3.10(5) oligodendrocytes per rat pup).

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1990

Effect of exogenous fatty acids on lipid synthesis, marker-enzymes, and development of glial cells maintained in serum-free culture.

Glial cells were isolated from the cerebra of 7-day-old rats and maintained in culture in a chemically defined medium that favours the development of oligodendrocytes. Acetate, butyrate, or albumin-bound hexanoate, octanoate, decanoate, laurate, myristate, palmitate, oleate, linoleate, or arachidonate was added to the culture medium. The incorporation of [3H]acetate into fatty acids and cholesterol and [35S]sulphate into sulphatide, and the activities of the oligodendrocyte marker enzymes 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase were measured. The composition of the glial cell population (the number of astrocytes and oligodendrocytes) in these cultures was studied by immunocytochemistry. Results show that 1) long-chain fatty acids depress the synthesis of fatty acids, cholesterol, and sulphatide; and 2) the presence of long-chain, in contrast to short-chain, fatty acids in the culture medium lowers the activities of 2',3'-cyclic-nucleotide 3'-phosphodiesterase and glycerol 3-phosphate dehydrogenase and decreases the number of oligodendrocytes. Our results suggest that long-chain fatty acids exert a negative influence on the development of oligodendrocytes in the culture system used.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1989

Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM-strain): studies in vivo and in vitro.

The pathogenicity and cell tropism of mouse hepatitis virus (MHV-JHM-strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus-infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV-JHM was examined further in virus-infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus-induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV-JHM.

Animals↗

PubMed1989

Development of oligodendrocytes. Studies of rat glial cells cultured in chemically-defined medium.

Oligodendrocytes are macroglial cells that synthesize and maintain myelin in the central nervous system. Oligodendrocytes in rodent brain are formed postnatally from glial progenitor cells. These progenitors cells are bipotential and differentiate in a later stage of development into type-2 astrocytes. Recent studies with cultured cells indicate that growth factors such as platelet-derived growth factor and ciliary neurotrophic factor are instrumental in the control of these events. This paper discusses various methods for the isolation of oligodendrocytes and for their maintenance in culture. We use cerebra or spinal cords from one-week old rat pups to prepare glial cultures that are enriched in oligodendrocytes (60-80% or greater than or equal to 90%, respectively). After one day in serum-containing medium the cells are kept in chemically-defined medium, supplemented with the hormones insulin, T3 and hydrocortisone. The activities of astrocyte-and oligodendrocyte-specific marker enzymes were measured to evaluate the influence of these hormones on the differentiation of the oligodendrocytes. Finally, glial energy metabolism and the utilization of ketone bodies and of fatty acids are discussed briefly.

Animals↗

PubMed1988

Effects of insulin and insulin-like growth factor (IGF-I) on oligodendrocyte-enriched glial cultures.

The brains from 1-week-old rat pups were used to prepare cultures of glial cells. After 24 hr in culture the cells were changed to a chemically defined serum-free medium (CDM). We have used antibodies against gangliosides (monoclonals A2B5 and LB1) and against galactocerebrosides to monitor the influence of insulin on the development of oligodendrocytes from precursor cells. In these oligodendrocyte-enriched cultures we investigated the influence of insulin and IGF-I on the activity of the marker enzymes 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and glycerol-3-phosphate dehydrogenase (G3PDH), and on lipid metabolism. 1) Incorporation of [35S] sulfate into sulfolipids was stimulated by insulin (optimal concentration 100 nM). A sharp peak in sulfolipid synthesis was seen at day 5-6 in culture. 2) Insulin stimulated the expression of CNPase and G3PDH in culture. 3) The stimulating effect of insulin on sulfolipid synthesis, CNPase, and G3PDH activity was mimicked by IGF-I (13 nM). 4) The incorporation of [35S] into sulfolipids and [2-3H]acetate into fatty acids and cholesterol was reversibly reduced by temporary omission of insulin from the medium. These results indicate that insulin and IGF-I stimulate the development of oligodendrocytes in our culture system and that insulin has a general anabolic effect on the lipid metabolism of the cells.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1988

Cellular location of glutamine synthetase and lactate dehydrogenase in oligodendrocyte-enriched cultures from rat brain.

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1987

Hydrocortisone stimulates the development of oligodendrocytes in primary glial cultures and affects glucose metabolism and lipid synthesis in these cultures.

Cultures of glial cells were prepared from the brains of one-week-old rat pups. After one day in culture, serum was omitted from the medium and replaced by a combination of growth-stimulating hormones and other factors that enhanced the percentage of oligodendrocytes in the cultures. We investigated the effects of hydrocortisone on the development of oligodendrocytes, on the activities of oligodendrocyte-specific enzymes and on glucose- and lipid-metabolism of the glial cells. Hydrocortisone greatly enhanced the survival of glial cells in culture. The development of galactocerebroside-positive cells and the specific activity of 2',3'-cyclic-nucleotide 3'-phosphodiesterase were stimulated by 50 nM hydrocortisone, whereas these effects were partly reversed at higher concentrations of the hormone. The specific activity of glycerol-3-phosphate dehydrogenase was markedly stimulated by hydrocortisone; 1 microM or higher concentrations of hydrocortisone were required for an optimal effect. The consumption of glucose and the production of lactate were lowered by hydrocortisone whereas the oxidation of [6-14C]glucose to 14CO2 was not affected. Incorporation of [35S]sulfate into sulfolipids was greatly enhanced by hydrocortisone and [14C]incorporation from [1-14C]acetate into cholesterol and fatty acids was also stimulated but to a smaller extent. These results show that hydrocortisone exerts a general trophic function on glial cells in our culture system; enhances the ratio of oligodendrocytes over astrocytes, possibly by directing bipotential progenitor cells to develop into oligodendrocytes; specifically induces glycerol-3-phosphate dehydrogenase in oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1986

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures.

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1986

Acetoacetate and glucose as lipid precursors and energy substrates in primary cultures of astrocytes and neurons from mouse cerebral cortex.

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.

3-Hydroxybutyric Acid↗

PubMed1984

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture.

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.

Acetoacetates↗

PubMed1984

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O.

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.

Acetoacetates↗

PubMed1984

Culture of rat cerebral oligodendrocytes in a serum-free, chemically defined medium.

Oligodendrocytes were isolated from the cerebra of young rats (5-10 days old) by trypsinization of the tissue followed by cell separation on Percoll gradients. The isolation was carried out in physiological, isotonic media. The cell yield was 2-4 X 10(6) cells per brain; the plating efficiency was greater than or equal to 70%. Isolated cells were seeded on poly-L-lysine-coated culture dishes and maintained in a serum-free, chemically defined medium for at least 30 days. After 10 days in culture 67 +/- 10% of the surviving cells were oligodendrocytes, as judged by immunocytochemical and morphological criteria, whereas most of the other cells reacted positively with antiserum against glial fibrillary acidic protein. The expression of typical oligodendrocyte markers (2':3'-cyclic-nucleotide 3'-phosphodiesterase, galactocerebrosides and myelin basic protein) was greatly enhanced under these serum-free conditions as compared with cultures in serum-containing medium. The antigenic markers (galactocerebrosides, myelin basic protein) were absent in the freshly isolated cells but could be detected after 3 days in culture by immunocytochemistry. The activity of 2':3'-cyclic-nucleotide 3'-phosphodiesterase increased from 75 nmol min-1 mg-1 protein on day 4 to 400 nmol min-1 mg-1 protein on day 14 in culture.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1983

Acetoacetate: a major substrate for the synthesis of cholesterol and fatty acids by isolated rat hepatocytes.

Evidence is presented that isolated, intact rat hepatocytes can synthesize fatty acids and cholesterol from acetoacetate. The quantitative importance of these processes is evaluated by measuring total rates of fatty acid and cholesterol synthesis by incorporation of 3H from 3H2O. The contribution of acetoacetate varies from 14-54% and from 21-75% for de novo synthesized fatty acids and cholesterol, respectively, depending on the physiological condition of the donor rat. The relative contribution of acetoacetate to cholesterol synthesis is 1.4-2.3-times greater than to fatty acid synthesis.

Acetoacetates↗

PubMed1983

Ketogenesis in mitochondria isolated from liver biopsies of normal and starved dogs: comparison with rat-liver mitochondria.

Fatty acid oxidation and ketogenesis were studied in isolated dog-liver mitochondria in order to investigate whether the absence of hyperketonemia in fasting dogs results from a low capacity of hepatic ketogenesis. Isolated rat-liver mitochondria were used as reference. The results indicate that: (a) Dog-liver mitochondria oxidize long-chain fatty acids and produce ketone bodies at about equal rates as rat-liver mitochondria. No differences were detected in the regulation of ketogenesis. (b) Rates of oxidation of medium-chain fatty acids are significantly lower in dog-liver mitochondria than in rat-liver mitochondria. (c) Fasting does not influence the capacity of liver mitochondria for fatty acid oxidation but their ketogenic capacity is slightly enhanced in both species. The regulation of the energy metabolism in the fasting dog is discussed and compared with that in other mammalian species.

Animals↗

PubMed1982

Ketone-body utilization by homogenates of adult rat brain.

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of 14C from [3-14C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that of D-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO2. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.

2,4-Dinitrophenol↗

PubMed1982

Citrate synthesis in intact rat-liver mitochondria is irreversible.

Rat-liver mitochondria were incubated with [1,5-14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion-exchange chromatography. Incorporation of 14C into acetyl-L-carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol . mg-1 min-1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg-1 . min-1. We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.

Acetyl Coenzyme A↗