Biomedical subjects
Publications and source records attributed to G Ramírez.
Magnetic resonance images were obtained from two isolated horses' heads. Ten mm thick, T1-weighted images were taken with a 1.5 Tesla magnet and a body coil, and compared with the corresponding frozen cross-sections of the heads, relevant structures being identified and labelled at each level. The images should provide reference material for clinical magnetic imaging studies of horses' heads.
Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.
Astrocytes and microglia are closely associated with amyloid plaques in Alzheimer's disease (AD). Microglia constitute the first barrier surrounding plaques, although they seem to be unable to remove them efficiently. We evaluated the reaction of microglial cells from neonatal rats and mice to plaque mimetics. The C-terminal part of the amyloid precursor protein (APP) or amyloid peptide (A beta) was immobilized to either 60-microm or 2.8-microm beads and incubated with microglial cells. Beads of 60 microm, having approximately the size of senile plaques, were not phagocytosed, in contrast to 2.8-microm beads, which were phagocytosed by microglia but not by astrocytes. Once taken up by the cells, proteins immobilized to the beads were degraded rapidly, as confirmed by mass spectrometry and immunofluorescence with an antibody against beta-amyloid. On the other hand, no protein degradation was observed with 60-microm beads. Also, probably as a reaction to its incapability to phagocytose the beads, glia organized around the beads and started to proliferate. Cell proliferation was more pronounced when the beads contained the A beta epitope compared with the beads with an inert surface. This in vitro effect could be exploited to set up a screening assay for compounds that ameliorate the adverse reaction of microglia supposed to contribute to the pathogenesis of AD.
AMPA (EC50 = 1.0 x 10(-6) M) and NMDA (EC50 = 1.3 x 10(-4) M) trigger 45Ca2+ influx in 13-day chick embryonic retinal explants. This agonist-driven cationic flux is specifically inhibited by typical competitive antagonists, such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-7-phosphonoheptanoate (AP7), respectively. Guanine nucleotides, with different degrees of phosphorylation, namely 5'-GMP, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and 5'-guanylyl-imidodiphosphate (GppNHp), are also efficient blockers of 45Ca2+ influx. These results confirm the antagonistic behavior of guanine nucleotides towards ionotropic glutamate receptors and suggest a convenient experimental approach for screening of novel agonists and antagonists.
In order to determine the temperature effect on the axial muscle growth of sea bass, a stock of larvae was subjected to the following incubation and cultivation temperatures, respectively: 15 degrees C/ambient, 15/17 degrees C, 17 degrees C/ambient and 17/17 degrees C. In all groups the cross-sectional area of white and red muscles and the number and average area of the white and red muscle fibres were quantified. Results showed that the embryonic period, pre-larval phase and the end of metamorphosis were accelerated at higher temperatures. During the endogenous feeding period, muscle growth took place by fibrillar hypertrophy, and was not influenced by the temperature. Thereafter (external feeding) muscular hyperplasia began, and growth of all the muscular parameters was favoured by the effect of high incubation and cultivation temperatures, with the latter having higher influence. High incubation temperature had an slight effect on muscle growth and body length, which was only observed from 15 days. Metamorphosis finished at 3 +/- 0.4 cm in all the larvae, but this length was earlier reached at higher temperatures. At 120 days, the largest growth was obtained in the larvae maintained at a higher temperature.
Neurofibromatosis is an autosomal dominant genetic disease, characterized by café au lait spots, neurofibromas and several bony anomalies. Deformities of the spine are the most frequent alterations. Involvement of the cervical spine has been studied less frequently. The case of a 16-year-old male patient affected by neurofibromatosis, with cervical pain without neurological symptoms is presented. X-rays, CT-scan and MRI demonstrated the presence of cervical kyphosis, occipitoaxial instability and atlantoaxial instability with subluxation. Posterior occipito-C2 fusion was performed with prior placement of a halo-vest. The outcome at four years was good with solid occipito axial fusion, moderate loss of cervical spine flexion and moderate-to-severe limitation of cervical spine rotation. The incidence and variety of alterations of the cervical spine in patients affected with neurofibromatosis is discussed, as well as the results obtained by the treatment.
Chick kainate binding protein was solubilized from cerebellar membranes and purified (x19) by use of two chromatographic steps. Measurements of [3H]kainate binding and GTPase activity in the different fractions reveal a consistent decrease of GTPase activity as the purification proceeds so that no GTPase is detectable after the final purification step. This fact, in the context of the differential involvement in nucleotide recognition of some critical amino acid residues in the p-loop motif of GTPases and in the guanine nucleotide-binding sequence of ionotropic glutamate receptors, together with significant discrepancies concerning the activity of individual nucleotides, suggests that both guanine nucleotide-recognizing sequences are unlikely to be alternative expressions of the same functional domain.
Guinea-pig spermatozoa in the presence of a purified fraction from Echeveria gibbiflora aqueous crude extract suffer a hypotonic-like effect. The phenomena exhibited included a distension of the plasma membrane over the acrosome region, inducing the formation of a huge 'head-bubble'. The agglutination effect was so enhanced that instead of inducing sperm clusters, it produced cane-like 'stalk' structures. The immobilizing activity was induced instantaneously after the addition of the purified fraction. At electron microscope level it was possible to observe a heavy amount of electron dense material of the purified fraction embedded or intercalated along the plasma membrane. It was also possible to corroborate the dispersion of the acrosomal content and the disappearance of the external acrosome membrane. The purified fraction induced loosening of the plasma membrane all along the sperm cell, however, the distension of the membrane was only produced in the apical portion of the sperm head and not in the post equatorial region. The results suggest that the plant may yield a compound suitable for use as a vaginal barrier or male contraceptive agent.
Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage-clamp conditions, induced dose-dependent (EC50 = 87+/-14 microM) inward currents, confirming the functional incorporation to the oocyte of kainate-driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA)-preferring receptors. Binding experiments with (S)-[3H]AMPA confirmed the presence in the chick membrane preparation of low-affinity AMPA receptors (K(D) = 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 microM kainate by approximately 90%. In the case of GMP, additional concentration-inhibition studies yielded an IC50 of 180+/-11 microM. Our results illustrate the apparent failure of kainate-binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand-binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms.
As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.
Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of guanine nucleotides (in the case of GMP and GDPbetaS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.
It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.
Metabotropic glutamate receptors (mGluRs) have been shown to modulate adenylate cyclase activity via G-proteins. In the present study we report similar results to the previously observed in the literature, showing that glutamate and the metabotropic agonists, 1S,3R-ACPD or quisqualate induced cAMP accumulation in hippocampal slices of young rats. Moreover, guanine nucleotides GTP, GDP or GMP, inhibited the glutamate-induced cAMP accumulation. By measuring LDH activity in the buffer surrounding the slices, we showed that the integrity of the slices was maintained, indicating that the effect of guanine nucleotides was extracellular. GMP, GDPbeta-S or Gpp(NH)p abolished quisqualate-induced cAMP accumulation. GDPbeta-S or Gpp(NH)p but not GMP inhibited 1S,3R-ACPD-induced cAMP accumulation. The response evoked by glutamate was also abolished by the mGluR antagonists: L-AP3 abolished glutamate-induced cAMP accumulation in a dose-dependent manner and MCPG was effective only at the 2 mM dose. DNQX was ineffective. We are reporting here, an inhibition induced by guanine nucleotides, via an extracellular site (s), similar to the observed with classical glutamate antagonists on a cellular response evoked by mGluR agonists.
Nerve cell dissociation has become a key procedural tool in the implementation of a number of techniques in cellular and molecular neurobiology. We report that a protease preparation from Streptomyces fradiae (henceforth SF-protease) dissociates viable and morphologically identifiable embryonic and mature neurons and glial cells from the central nervous system of chick and rat, when used under strictly controlled conditions. Typical dendritic and axonal growth cones, with their lamellipodia and filopodia, are seen in many neuroblast types - growth cones in the case of embryonic glial cells and even the thinnest processes of some cells, such as the microvilli of adult chick retinal Müller (glial) cells, or the cilia of photoreceptors appear intact. Our results suggest that the SF-protease releases cells from tissue in a way that ensures the continuity of the plasma membrane and cuts through the transmembrane attachment systems (either cell-cell or cell-extracellular matrix) without compromising the cytoskeletal integrity underlying native cell shape.
When injected into the rat striatum, quinolinic acid causes dose-dependent widespread cell death. All cell types, including the NADPH-diaphorase-positive neurons appear to be sensitive to the toxin. The latter cells are destroyed by quinolinic acid injections of 180 nmol per striatum, this effect being blocked by the concomitant administration of 5 mg/kg of the non-competitive N-methyl-D-aspartate antagonist MK-801. We report that guanosine-5'-monophosphate (GMP), at a dose of 360 nmol, is equally effective in protecting the diaphorase-positive cells against quinolinate toxicity.
Guanine nucleotides (GNs), including GMP, displace [3H]kainic acid binding to chick cerebellar lysed and vesiculated membranes. Saturation studies of [3H]GppNHp binding, under conditions that prevent the occupation of the nucleotide binding sites in G-proteins, demonstrate the existence of extracellular membrane receptors specific for guanine nucleotides. Affinity-labeling of a vesicle preparation with [alpha-32P]GTP gives one single labeled band, upon electrophoresis, with an apparent molecular mass of 50 kDa. Additional experiments with partially purified kainate receptors suggest that the GN extracellular sites may overlap, at least partially, the kainic acid binding sites, being then responsible for the displacement of [3H]kainic acid by GNs. The physiological significance of these findings remains unclear.
OBJECTIVES: To examine the overall effectiveness of patient education and counseling on preventive health behaviors and to examine the effects of various approaches for modifying specific types of behaviors. DATA SOURCES: Computerized databases (Medline, Healthline, Dissertation Abstracts, and Psychological Abstracts), bibliographies 1971-1994. Search terms (patient education, patient compliance, and self care) with modifiers (evaluation and specific preventive behaviors). STUDY SELECTION: Randomized and non-randomized controlled trials measuring behavior in clinical settings with patients without diagnosed disease. Abstracts and retrieved studies screened by multiple reviewers; 13% of retrieved studies met screening criteria. DATA EXTRACTION: Replicated coding by multiple observers. DATA SYNTHESIS: Behaviors were grouped based on whether the behavior is addictive and whether the desired change required subtraction of existing behaviors or adding new behaviors. The weighted average effect size from a random effects model for smoking/alcohol studies was 0.61 (CI = 0.45, 0.77), for nutrition/weight, 0.51 (CI = 0.20, 0.82) and for other behaviors, 0.56 (CI = 0.34, 0.77) indicating that the behavioral outcomes for these subgroups were significantly different from zero. Multiple regression models for the three groups indicated that using behavioral techniques, particularly self-monitoring, and using several communication channels, e.g., media plus personal communication, produces larger effects for the smoking/alcohol and nutrition/weight groups. CONCLUSIONS: Patient education and counseling contribute to behavior change for primary prevention of disease. Some techniques are more effective than others in changing specific behaviors.
OBJECTIVES: To review the diagnostic utility of the chest radiograph for left ventricular dysfunction. DATA SOURCES: Structured MEDLINE searches, citation reviews of relevant primary research, review articles, and textbooks, personal files, and data from experts. STUDY SELECTION: Studies of patients without valvular disease that allowed calculation of the sensitivity and specificity of selected radiographic signs compared with a criterion standard of increased left ventricular preload or reduced ejection fraction. DATA EXTRACTION: Two independent readers reviewed 29 studies. Studies were pooled after stratification by radiographic finding, criterion standard, and clinical setting. MAIN RESULTS: Redistribution best diagnosed increased preload with a sensitivity of 65% (95% confidence interval [CI] 55%, 75%) and specificity 67% (95% CI 53%, 79%). Cardiomegaly best diagnosed decreased ejection fraction with a sensitivity of of 51% (95% CI 43%, 60%) and specificity of 79% (95% CI 71%, 85%). Interrater reliability was fair to moderate for redistribution and moderate for cardiomegaly. The clinical setting affected results by decreasing the specificity of cardiomegaly to 8% in detecting increased preload in patients with severe systolic dysfunction. The absence of redistribution could only exclude increased preload in situations in which the suspicion (pretest probability) of disease was less than 9%, whereas redistribution could confirm increased preload when the pretest probability was greater than 91%. The absence of cardiomegaly could only exclude a reduced ejection fraction if the pretest probability was less than 8%, whereas cardiomegaly could confirm a reduced ejection fraction if the pretest probability was greater than 87%. CONCLUSIONS: Redistribution and cardiomegaly are the best chest radiographic findings for diagnosing increased preload and reduced ejection fraction, respectively. Unfortunately, neither finding alone can adequately exclude or confirm left ventricular dysfunction in usual clinical settings. Redistribution is not always reliably interpreted.