Juan Manuel de Gandarias

Effects of fluoxetine administration on mu-opoid receptor immunostaining in the rat forebrain

Fluoxetine is a selective serotonin reuptake inhibitor. Analysis of mu-opioid receptor immunostaining after chronic fluoxetine administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic fluoxetine treatment.

Polyploidization and exit from cell cycle as mechanisms of cultured melanoma cell resistance to methotrexate

Numerous malignant neoplasias are found to contain varying proportions of high-ploidy cells. Although the role they play in the tumor is poorly understood, several lines of evidence suggest that these cells could be especially resistant to various aggressions, a possibility of great interest in cancer treatment. In the present study, we tested this hypothesis through the analysis of the presence of high-ploidy cells following the administration of the chemotherapeutic agent methotrexate. We also determined the expression of two proliferation markers, PCNA and CDK1, after methotrexate-treatment. Cultured cells from the murine melanoma B16F10 were treated with high doses of methotrexate for seven days prior to determination of DNA content and proliferation markers. Our results showed an obvious increase in the mean ploidy of this population. Specifically, there was a dramatic reduction in the proportion of tetraploid cells (predominant in the original population), and an increase in the proportion of cells with higher ploidies, particularly those whose DNA content was greater than 8c, including some cells with ploidies greater than 16c. Furthermore, there was a reduction in the number of PCNA-expressing cells and the reduction was much more marked in the case of CDK1 that was almost absent in the modal-ploidy treated cells. These alterations concerning ploidy and expression of proliferation markers had completely reverted two weeks after withdrawal of the drug. Our results indicate that methotrexate at a high dosage selects a cell population heterogeneous concerning its ploidy level, composed of one subpopulation of high-ploidy cells and another of modal-ploidy cells that, considering its lack of CDK1 expression, would remain in a latent state to evade the effects of the drug.

Mn2+-activated aspartat aminopeptidase activity, subcellular localization in young and adult rat brain

The levels of soluble and membrane-bound aminopeptidase activities were assayed in subcellular fractions from young (4 weeks old) and adult (20 weeks old) rat brains, using Asp-2-naphthylamide as substrate. The young rats showed the highest soluble and membrane-bound levels of activity in the microsomal fraction but no differences among fractions were found at 20 weeks of age. The membrane-bound activity was significantly higher than the soluble one in all subcellular fractions of young rats. Soluble activity of the homogenate and the mitochondrial fraction was significantly increased in adult animals when compared to that of younger ones.

The binding of thioureylene compounds to human serum albumin

The binding interactions of some thioureylene compounds to human serum albumin were studied in vitro by ultraviolet spectroscopy and equilibrium dialysis. Binding of 6-n-propyl-2-thiouracil, 6-n-benzyl-2-thiouracil and 2-thiouracil to human serum albumin results in a red shift of the ultraviolet absorption maximum, suggesting that the binding site is a hydrophobic area of the protein. Bindings of 6-n-propyl-2-thiouracil and 6-n-benzyl-2-thiouracil to human serum albumin are characterized by two classes of sites while 6-n-propyl-uracil and 2-thiouracil bind to one low-affinity binding site. In addition, an identification of those sites was performed by measuring the displacement of these drugs. The data show that the moderate-affinity site is common with the warfarin site while the low-affinity site is likely to be shared by benzodiazepines. It is concluded that the binding is enhanced by the hydrophobicity of the substituent in the thioureylene compounds, and it is further shown that thiol-group substitutions in the thioureylene ring will weaken the binding.

Daily Rhythm of Aspartate Aminopeptidase Activity in the Retina, Pineal Gland and Occipital Cortex of the Rat

The levels of specific soluble aspartyl aminopeptidase activity were assayed in retina, occipital cortex, anterior pituitary, posterior pituitary, pineal gland and serum of adult male rats, using Asp-2-naphthylamide as substrate, in a 12:12 h light:dark cycle (7-19 h light). Significant diurnal variations appeared in retina, pineal gland, occipital cortex and serum. In addition, different patterns of diurnal variation of the enzymatic activity were observed in the tissues analyzed. A regular increase of the activity was noticed at the end of the dark period in all the tissues as a common feature, except in serum, in which the enzymatic activity reached a peak in the middle of the light period, decreasing progressively during the dark hours. After the last hours of darkness, the pattern of variation in the activity differed in each tissue. These diurnal variations in aspartyl aminopeptidase activity could reflect the functional status of its putative endogenous substrates, such as angiotensin II, and it may also suggest the existence of differential regulatory mechanisms associated with each location.

Effect of imipramine on enkephalin-degrading peptidases

In recent years, there has been increasing evidence of the involvement of the endogenous opioid system in mental depression and its treatment. In this work, we have measured the effect of imipramine on enkephalin-degrading peptidases in several rat brain areas. Aminopeptidase activities have been assayed using Tyr-beta-naphthylamide as substrate and puromycin as selective inhibitor. Dansyl-D-Ala-Gly-Phe(pNO2)-Gly has been the substrate for neutral endopeptidase 24.11. Imipramine in vitro inhibits puromycin-sensitive activities in all brain areas studied, without affecting the rest of the enzymes assayed. However, subacute imipramine treatment increases neutral endopeptidase activity in the hypothalamus and chronic treatment increases this activity in the hypothalamus and the striatum. These results suggest to us that enkephalin-degrading peptidases are involved in the acute and chronic action mechanism of imipramine and reinforce the idea that the central enkephalinergic activity is dynamically changed during the treatment of depressive illness.

Subcellular ontogeny of brain pyroglutamyl peptidase I.

The present work studies pyroglutamyl-peptidase I activity in several subcellular fractions of the developing brain. In the synaptosomal fraction, soluble pGlu-peptidase I activity is low until postnatal Day 9 (with a peak at postnatal Day 2) and the activity increases at postnatal Day 15. In later stages there are not significant changes of synaptosomal pGlu-peptidase I activity. However, in the cytosolic fraction the activity is high from ED22 until postnatal Day 9, and afterwards decreases. The changes in the particulate fractions are generally less drastic.

Ontogeny of puromycin-sensitive and insensitive aminopeptidase activities in several subcellular fractions of the rat brain

Puromycin-sensitive and insensitive aminopeptidase (aminopeptidase M) activities are measured in several subcellular fractions of the rat brain cortex and subcortex during the first postnatal month. Tyr-beta-naphthylamide has been used as substrate and 20 microM puromycin as selective inhibitor. We have found that puromycin-sensitive aminopeptidase activity increases twofold in the synaptosomal and mitochondrial fractions in the first 6-9 postnatal days, just during the period of axonal and dendritic growth. This enzyme also has significant age-related changes in the nuclear fraction. The developmental pattern is different, depending on the subcellular fraction analyzed. Significant developmental changes of puromycin-insensitive aminopeptidase (aminopeptidase M) are only found in the myelinic and microsomal fractions and they are less significant than those found in the puromycin-sensitive aminopeptidase. It has been suggested that these enzyme activities could be involved in processes of cell proliferation, differentiation, and maturation.

Subcellular analysis of Tyr-aminopeptidase activities in the developing rat cerebellum

The endogenous opioid system seems to play important roles in the developing cerebellum. The first opioid peptide isolated, Met-enkephalin, is expressed transiently in this brain area. In the present study, several enzyme activities capable of hydrolyzing enkephalins are measured during the first month of cerebellar development, using Tyr-beta-naphthylamyde as substrate and puromycin as inhibitor of one of the membrane-bound aminopeptidases. Puromycin-sensitive soluble and membrane-bound aminopeptidase activities decrease in the synaptosomal and mitochondrial fractions at the end of the first month of life, just when enkephalin-like immunoreactivity decreases in the cerebellum. Membrane-bound enzyme also decreases in the myelinic fraction. Synaptosomal activity increases after birth, coinciding with decreases in the activity in the microsomal fraction. Puromycin-insensitive and membrane-bound aminopeptidase shows less significant developmental changes and they occur mainly in the first week of life, coinciding with the axonal and dendrite growth. These results could suggest a possible role of these enzymes, together with the rest of the opioid system, in cerebellar development.

Effect of subacute toluene administration on the enkephalinergic neuromodulatory system in rats and protective action of ganglioside treatments.

Gangliosides perform protective functions in the central nervous system. This paper describes a study of the effect of ganglioside administration on toluene neurotoxicity. Rat brain met-enkephalin immunostaining in the central amygdaloid nuclei showed changes in rats treated simultaneously with gangliosides and toluene with respect to rats treated with toluene alone. It is suggested that gangliosides prevent toluene neurotoxicity at this level, leading to hypothetical neurobehavioral changes.