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Biomedical subjects

A Aranda

Publications and source records attributed to A Aranda.

At least 91 records · Page 5Linked to original sources
PubMed1988

Triiodothyronine (T3) induces neurite formation and increases synthesis of a protein related to MAP 1B in cultured cells of neuronal origin.

Neuroblastoma (N2A) cells were found to develop axon-like neurite extensions when grown in the presence of triiodothyronine (T3), while C6 cells (of glial origin) did not. Analysis of radiolabelled protein synthesis showed that, in N2A only, T3 increased the synthesis of a polypeptide corresponding in electrophoretic mobility to the microtubule-associated protein MAP 1B. Immunoblotting of total cell proteins with a monoclonal antibody confirmed that this polypeptide was immunologically related to MAP 1B. Further studies using indirect immunofluorescence with monoclonal antibodies against both tubulin and MAP 1B showed that both antigens were present in neurites. Taken together, these results suggest that T3 may control maturation of neural tissue via effects on the microtubule-associated proteins in cells of neuronal origin.

Animals↗
PubMed1988

Mechanism of L-triiodothyronine (T3) uptake by glial C6 cells: regulation by butyrate.

The mode of entry of triiodothyronine (T3) and its regulation by butyrate was studied in cultured glial C6 cells. Uptake of [125I]T3 increases for at least 60 min in C6 cells. The amount of cell-associated radioactivity is 2- to 4-fold higher during the entire time-course in cells previously exposed to 2 mM butyrate for 48 h. Uptake was non-saturable since uptake velocity was linearly related to the extracellular hormone concentration between 0.2 and 800 nM T3 in control and butyrate-treated cells. Uptake velocity increased by more than 3-fold in the cells incubated with the fatty acid. T3 uptake was temperature dependent and the effect of butyrate was observed at the different temperatures examined. Preincubation with metabolic inhibitors did not block [125I]T3 uptake in either group, and monodansylcadaverine was also ineffective. Present results suggest that in C6 cells T3 uptake proceeds by a passive, energy-independent, non-saturable process, that is markedly affected by short-chain fatty acids. Additionally, this is the first study documenting that a natural compound directly influences the entry of thyroid hormones into cells.

Animals↗
PubMed1988

Comparison of the effects of forskolin and dibutyryl cyclic AMP in neuroblastoma cells: evidence that some of the actions of dibutyryl cyclic AMP are mediated by butyrate.

We have compared the effects of forskolin, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP, Bt2-cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2-cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose-dependent inhibition of cell proliferation, but the effect of Bt2-cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2-cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2-cAMP than in untreated cells or in forskolin-treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2-cAMP produced histone acetylation, a well-known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2-cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1(0), a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1(0) in the cells treated with Bt2-cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2-cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.

Acetylation↗
PubMed1988

Fibrinolytic activity in plasma after gynecological and urological surgery.

Fibrinolytic activity was studied in 51 patients subjected to major surgery (27 urological and 24 gynecological) without prophylaxis. Postoperative deep-vein thrombosis (DVT) was diagnosed by venography in 6 patients (11%). Blood samples were obtained preoperatively and on days 1, 3 and 7 postoperatively. Reduced fibrinolytic activity, as determined by prolongation of euglobulin lysis time (ELT), decrease in plasminogen activators on fibrin plate and decrease in tissue-type plasminogen activator (t-PA) activity were present postoperatively. Moreover, a marked increase in short duration in fast-acting inhibitor (PA-inhibitor) levels was observed on postoperative day 1 (p less than 0.001). Patients with DVT had higher fibrinogen degradation product (FDP) levels on postoperative days 3 and 7 (p less than 0.02). Preoperative PA-inhibitor activity was higher (p less than 0.02) in those patients who developed postoperative DVT. It is concluded that the decrease in t-PA and the increase in PA-inhibitor may contribute to the reduced postoperative fibrinolytic activity after major surgery. PA-inhibitor may represent a useful predictive marker of postoperative DVT.

Adult↗
PubMed1988

Evidence for the presence of nuclear 3,5,3'-triiodothyronine receptors in secondary cultures of pure rat oligodendrocytes.

It has been reported that oligodendrocytes do not contain nuclear T3 receptors, which is in apparent contradiction with the well-known effects of thyroid hormones on myelination. In this study we have reexamined the presence of receptors in this cell population, using pure rat oligodendrocyte cultures. T3 binding was also studied with the use of pure rat astrocytes as well as in mixed neuronal-glial cultures. The latter are mainly neuronal during the first days in culture and essentially glial thereafter. Binding studies carried out in intact cells demonstrated the presence of high affinity-low capacity binding sites for thyroid hormones in pure cultures of oligodendrocytes. The maximal binding capacity was 50-60 fmol/100 micrograms DNA and the dissociation constant (Kd) 0.13 nM. Pure rat astrocyte cultures also contained high affinity sites for thyroid hormones, although receptor concentrations was 2-3 times lower than in oligodendrocytes or neurons. This was confirmed in pure cultures of chick astrocytes and in neuronal-glial cultures during the astroglial period. The relative affinity of the receptor for thyroid hormone analogs was triiodothyroacetic acid = T3 greater than T4 greater than tetraiodothyroacetic acid in oligodendrocyte and astrocyte nuclei, and the sedimentation coefficient of the receptor was approximately 3.8S in both cell types. These results demonstrate that nuclear T3 receptors similar to those found in neurons and astrocytes are also present in oligodendrocytes. This suggests that the effects of thyroid hormones on myelination could result from a direct action of the hormone in the oligodendrocytes.

Animals↗
PubMed1988

Short chain fatty acids modulate nuclear receptor and extranuclear L-triiodothyronine levels in glial C6 cells by different mechanisms.

After incubation with [125]T3, the amount of radioactivity associated with the nuclear receptor and extranuclear compartment increases in glial C6 cells previously exposed to millimolar concentrations of butyrate. These actions of the fatty acid are accompanied by an inhibition of cell division, as shown by the decrease in [3H]thymidine incorporation or total DNA content per culture. Isobutyrate produced a similar effect at concentrations higher than 2 mM, whereas between 0.5-2 mM it increased the receptor content without affecting either extra-nuclear hormone or DNA. For both fatty acids there was a close parallelism between the dose-response curve for the inhibition of turnover of [3H]acetate from the histones and the increase in receptor levels. In contrast, a different dose dependence was found for the increase in extranuclear T3 levels, which suggests that the latter is caused by a mechanism other than histone acetylation. The increase in extranuclear T3 levels correlated better with the decrease in DNA and the accumulation of the chromosomal protein H10, which is thought to be related to the inhibition of cell proliferation. This suggests a relationship between the increase in extranuclear T3 and the inhibition of DNA synthesis. However, other agents, such as glucocorticoids or cAMP, which also inhibit C6 cell proliferation, were ineffective in increasing not only the receptor but also the extranuclear hormone. These findings show that the effect of short chain fatty acids on the receptor is not a consequence of the inhibition of cell replication, and that this inhibition could be related to, but is not sufficient per se for causing the increase in extranuclear T3.

Acetates↗
PubMed1987

Down-regulation of thyroid hormone nuclear receptor levels by L-triiodothyronine in cultured glial C6 cells.

L-Triiodothyronine (T3) produced a time- and dose-dependent depletion of nuclear thyroid hormone receptor levels in C6 cells, a rat glioma cell line. Receptor number diminished by 30-40% after a 48 h incubation with concentrations of T3 that saturate the nuclear receptor. The nuclear binding curve obtained in cells incubated for 48 h with T3 was shifted leftward of the curve obtained after a 3 h incubation, which indicates an apparent increase in receptor affinity after long-term incubation with T3. However, this change probably represents a further equilibration of the hormone, since the dissociation rate from the nuclei was similar in C6 cells after long- and short-term incubation with T3. The effect of T3 was further demonstrated in C6 cells incubated with short-chain fatty acids. Butyrate and isobutyrate increased receptor levels, and T3 partially decreased the response to these compounds. These findings suggest the existence of a desensitization process by which C6 glial cells would be protected against an excess of thyroid hormone.

Animals↗
PubMed1987

Effects of iopanoic acid on thyroid hormone receptor, growth hormone production, and triiodothyronine generation from thyroxine in pituitary GH1 cells.

We have studied the effect of iopanoic acid (IOP), a radiographic contrast agent which inhibits T4 to T3 conversion, on thyroid hormone nuclear receptors, GH response to T4 and T3, and T4 5'-monodeiodination in GH1 cells, a rat pituitary cell line. IOP at concentrations higher than 10 microM inhibits iodothyronine binding to the nuclear receptor without changing the dissociation constant (Kd) (0.1 nM for T3 and 1 nM for T4), and reduces the GH response to 50 nM T4, 5 nM T3, or the combined effect of T4 and glucocorticoids. These results could be explained by an inhibition of protein synthesis which was reduced by more than 50% by 50 microM IOP. By contrast, nontoxic concentrations of IOP did not change the GH response to different doses of T4 ranging from 1 nM to 50 nM. We also examined T3 generation from T4 and found that the intracellular T3 levels of cells incubated with 50 nM T4 were almost as high as those of cells incubated with 5 nM T3 which induces a full GH response. Intracellular T3 levels were markedly reduced in the cells incubated with T4 and IOP, but GH production was not reduced despite these differences in T3 levels. Additionally, more than 40% of the nuclear receptor was occupied by T3 in cells incubated with T4, whereas more than 90% was occupied by T4 in cells receiving the same amount of T4 with 5 microM IOP. Our results suggest that the effect of T4 on GH production by GH1 cells could be attributed to an important extent to the T3 generated from it, whereas when T4 monodeiodination is strongly inhibited, most of the biological activity is a result of intrinsic T4 activity.

Animals↗
PubMed1987

Glucocorticoids regulate insulin binding in a rat glial cell line.

We have examined insulin receptor regulation by glucocorticoids in the C6 rat glioma cell line. Dexamethasone decreased insulin binding to intact cell monolayers in a dose and time-dependent fashion. The effect is maximal between 48 and 72 h with 50 nM dexamethasone that decreased binding by 40-60%. The natural steroid corticosterone produced a similar effect although it was less potent, and the antiglucocorticoid 17 alpha-methyltestosterone was ineffective in lowering the receptor and partially antagonized the effect of dexamethasone. Total number of binding sites was decreased by glucocorticoids, and when analyzed with a two-site model a 3-fold increase in the dissociation constant (Kd) of the low affinity site was also observed. In the absence of protein synthesis the receptor accumulates at the cell surface, since cycloheximide produced a large increase of insulin binding. Cycloheximide totally blocked the effect of dexamethasone when both compounds were added together to the cells suggesting that protein synthesis is necessary for the effect of the glucocorticoid. By contrast, in cells pretreated with dexamethasone, cycloheximide was unable to produce an increase in cell surface receptor, showing that glucocorticoids probably deplete not only membrane receptor but also total cellular receptor.

Animals↗
PubMed1986

Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation.

We have studied the effect of butyrate and other short-chain fatty acids on thyroid hormone nuclear receptors in C6 cells, a rat glioma cell line. Exposure of C6 cells to butyrate leads to increased levels of L-triiodothyronine (T3) in the nuclear and extranuclear compartments. The rise in nuclear binding is not merely a reflection of the higher cellular hormone content, and Scatchard analysis of T3 binding to isolated nuclei reveals that butyrate increases receptor number without changing affinity. The effect on the receptor is quantitatively important: a 48-h incubation with 2 mM butyrate increases nuclear binding by 2-3-fold, and 5 mM butyrate by 3-5-fold. Other short-chain fatty acids were found to similarly influence both nuclear receptor and extranuclear T3 levels with the following potency: butyrate greater than valerate greater than propionate greater than acetate. On the contrary, ketone bodies were ineffective. Butyrate increases receptor levels by decreasing receptor degradation, since the apparent t1/2 of receptor disappearance increased by approximately 3-fold in cells incubated with 2 mM butyrate for 48 h. The regulation of receptor number might be secondary to an action of butyrate on regions of the chromatin to which the receptor associates. We then examined the effect of butyrate on histone acetylation. The fatty acid had little effect in increasing the level of multiacetylated forms of H3 and H4 histone when studied in acid-urea gels, but it markedly inhibited the turnover of [3H] acetate from the histone fraction. There was a striking similarity in the dose-response of butyrate for increasing receptor levels and inhibiting histone deacetylation. Furthermore, a very close correlation between receptor levels and [3H]acetate release was also found when different short-chain fatty acids were used. We thus conclude that the effect of butyrate on the receptor could be explained by a modification of the chromatin structure of C6 cells secondary to acetylation.

Acetates↗
PubMed1986

Regulation of glycerol phosphate dehydrogenase and lactate dehydrogenase activity by forskolin and dibutyryl cyclic AMP in the C6 glial cells.

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.

Animals↗
PubMed1986

Respiratory systems of the Bacillus cereus mother cell and forespore.

The respiratory systems of the mother cells and forespores of Bacillus cereus were compared throughout the maturation stages (III to VI) of sporulation. The results indicated that both cell compartments contain the same assortment of oxidoreductases and cytochromes. However membrane fractions from young forespores were clearly distinct from those of the mother cell, i.e., lower content of cytochrome aa3, lower cytochrome c oxidase activity, higher concentration of cytochrome o, and a lower sensitivity of the respiration to the inhibiting effect of cyanide. This suggests that the cyanide-resistant pathway contributes more importantly to forespore respiratory activity than to activity in the mother cell compartment. During the maturation stages, the forespore NADH oxidase activity declined faster than in the mother cells. Other activities studied decreased steadily in both cell compartments. These findings together with the analysis of the kinetics of NADH-dependent reduction of cytochromes in the mature spore membranes indicated an impairment of electron flow between NADH dehydrogenase and cytochrome b. This impairment could be overcome by the addition of menadione.

Bacillus cereus↗
PubMed1986

Triiodothyronine receptors in developing mouse neuronal and glial cell cultures and in chick-cultured neurones and astrocytes.

The evolution of L-triiodothyronine (T3) receptors was studied in developing cultures of cells dissociated from cerebral hemispheres of 14-day-old mouse embryos, which present successive distinct periods of cell proliferation and/or maturation. These periods are characterized essentially as neuronal from 1 to 12 days in vitro (DIV) and glial between 12 and 60 DIV. Furthermore myelin-related membranes are produced in this culture system. Binding capacities of the T3 nuclear receptors increased from 1 to 6 DIV, when it reached a maximum (16 fmol/100 micrograms DNA). A similar increase of the DNA content of the cell was observed until 8 DIV. Thereafter a sharp fall of receptor concentration leading to a 5-fold decrease in the binding capacity occurred until day 15, a period at which neurones disappeared from the cultures. From 25 to 50 DIV (coinciding with the glial period), the concentration of receptor remained more or less constant (1-2 fmol/100 micrograms DNA). In parallel, the DNA content did not vary greatly between 30 and 50 DIV. Scatchard analysis revealed the presence of a single class of receptors at 6 and 20 DIV, representative of 'neuronal' and 'glial' periods, respectively. The equilibrium dissociation constant (Kd) of the nuclear receptor from cells at 6 DIV (2 X 10(-10) M) was similar to that found at 20 DIV. These results were confirmed using pure cultured neurones and astrocytes prepared from embryonic chick brain. The effect of T3 on the cellular gangliosides used as an index of neuronal cell maturation, and on cerebroside sulfotransferase (CST), an enzyme involved in the production of myelin sulfatides, was studied to determine a possible correlation between the binding capacity of the T3 nuclear receptor and the response of the cultured cells to thyroid hormone. Our data demonstrate that T3 had no significant effect either on the content of gangliosides or on their developmental pattern, while it increased the level of CST activity by 75% between 18 and 25 DIV. These results show that, although the concentration of T3 receptors per 100 micrograms DNA in glial cells was lower than that in neurones, it was nevertheless sufficient to elicit a response in oligodendrocytes.

Animals↗
PubMed1986

Identification and characterization of L-triiodothyronine receptors in cells of glial and neuronal origin.

High affinity-low capacity binding sites for thyroid hormone have been identified in the nuclei of glial (C6) and neuronal (Neuro 2A) cultured cells. Equilibrium dissociation constants, determined by Scatchard analysis, were very similar in both types of cells (0.2-0.3 nM). The relative affinity of hormonal analogs was also similar: the affinity for T3 was lower than for triiodothyroacetic acid and higher than for T4 or tetraiodothyroacetic acid. The sedimentation coefficients obtained by gradient centrifugation of nuclear receptor extracted with 0.4 M KCl or excised by micrococcal nuclease digestion were 3.5 S and 6.5 S, respectively. These results suggest that the thyroid hormone receptor is not restricted to neuronal cells, but also appears in cells of glial origin.

Animals↗
PubMed1984

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells.

The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells.

Animals↗