Palmitoylation of nicotinic acetylcholine receptors.

It is well established that nicotinic acetylcholine receptors (nAChRs) undergo a number of different posttranslational modifications, such as disulfide bond formation, glycosylation, and phosphorylation. Recently, our laboratory has developed more sensitive assays of protein palmitoylation that have allowed us and others to detect the palmitoylation of relatively low abundant proteins such as ligand-gated ion channels. Here, we present evidence that palmitoylation is prevalent on many subunits of different nAChR subtypes, both muscle-type nAChRs and the neuronal "alpha(4)beta(2)" and "alpha(7)" subtypes most abundant in brain. The loss of ligand binding sites that occurs when palmitoylation is blocked with the inhibitor bromopalmitate suggests that palmitoylation of alpha(4)beta(2) and alpha(7) subtypes occurs during subunit assembly and regulates the formation of ligand binding sites. However, additional experiments are needed to test whether nAChR subunit palmitoylation is involved in other aspects of nAChR trafficking or whether palmitoylation regulates nAChR function. Further investigation would be aided by identifying the sites of palmitoylation on the subunits, and here we propose a mass spectrometry strategy for identification of these sites.

[Cholesterol crystals pericarditis]. / Pericarditis con cristales de colesterol.

Cholesterol pericarditis is an uncommon, specific form of pericardial disease that is characterized by the presence of cholesterol crystals in pericardial fluid. The etiology may be either idiopathic or in association with systemic disorders such as tuberculosis, rheumatoid arthritis, myxedema or hypercholesterolemia. We report a case of cholesterol pericarditis in a 51-year-old male who was diagnosed with chronic renal failure due to polycystic kidney disease. To our knowledge no similar cases have been reported to date in the literature.

The unusual nature of epibatidine responses at the alpha4beta2 nicotinic acetylcholine receptor.

The identification of an equatorial frog toxin, epibatidine, as a potent non-morphinic analgesic, selective for neuronal nicotinic acetylcholine receptors, provoked a marked renewal in our understanding of pain and its mechanisms. In this work we have examined the effects of epibatidine at the major brain rat alpha4beta2 nicotinic acetylcholine receptor expressed in a cell line. Fast drug applications obtained with a modified liquid filament system were used for the analyses of the currents evoked by acetylcholine, nicotine and epibatidine. Characterized by a slow onset and offset, epibatidine responses were of smaller amplitude to those evoked by acetylcholine or nicotine. About a thousand times more sensitive to epibatidine than acetylcholine, the alpha4beta2 receptor also displayed a more pronounced apparent desensitization to this compound. Finally, overnight exposure to 1 nM epibatidine failed to produce the functional upregulation observed with nicotine. These data indicate that, at the rat alpha4beta2 receptor, epibatidine acts as a partial agonist causing a pronounced inhibition of agonist evoked currents at concentrations that do not activate the receptors.

alpha-bungarotoxin receptors contain alpha7 subunits in two different disulfide-bonded conformations.

Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.

Evaluación ultrasonográfica de las mediciones renales en el neonato de término / Ultrasound evaluation of renal measures in normal full term newborn infant

Objetivo. Presentar la distribución de los valores en la medición renal en recién nacidos a término, sin patología agregada, bajo la posible influencia de las variables de edad gestacional, peso al nacer y sexo. Material y métodos. Se incluyeron a recién nacidos a término, sin patología agregada; fueron resgistradas las variables de edad gestacional, sexo y peso al nacer, evaluadas en los primeros tres días de vida. Se realizó ultrasonido abdominal, empleando un transductor sectorial multifrecuencia de 7.5 MHz. Se efectuaron las mediciones longitudinal y anteroposterior, así como el cálculo de perímetro y el área de ambos riñones. Resultados. Se incluyeron 83 neonatos. No se observaron diferencias significativas en las mediciones ultrasonográficas entre ambos riñones, ni con respecto a las variables de sexo, edad gestacional o peso al nacer. Los valores promedios de toda la muestra, fueron de 4.1, 2.0, 10.3 cm y 6.8 cm2, para las mediciones longitudinal, diámetro anteroposterior, perímetro y área, respectivamente. Se reportaron los valores percetilares de cada medición. Conclusiones. No se documentó el efecto de las varibles de sexo, edad gestacional o peso al nacimiento sobre mediciones renales; estos resultados pueden ser útiles como referencia en la evaluación de la morfología y tamaño de la silueta renal en la población neonatal de recién nacidos de término

Transient expression of heteromeric ion channels.

Transient transfection is an excellent method for the expression and study of cell-surface, heteromeric ion channels. The cell type, the total amount of DNA, the combination of subunits and the ratio of subunit DNA are all important parameters to consider when attempting to optimize expression. A serious drawback of this method is that the efficiency of subunit assembly is very low in comparison to the efficiency of assembly for stably expressed heteromeric ion channels. The low efficiency of assembly prevents use of transient expression methods for detailed studies of heteromeric AChR assembly, and caution should be taken in the use of these methods for the study of intracellular heteromeric ion channel subunits. After the transient expression of heteromeric AChR subunits, virtually all of the expressing cells contained all four AChR subunits. However, the subunits were heterogeneously distributed among the cells, and the low efficiency of AChR assembly appears to be due to cell-to-cell variations in the ratio of the four subunits.

A conserved role for L1 as a transmembrane link between neuronal adhesion and membrane cytoskeleton assembly.

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, we demonstrate here that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations results from a disruption of the link between the extracellular environment and the neuronal cytoskeleton.

Ethanol does not inhibit the adhesive activity of Drosophila neuroglian or human L1 in Drosophila S2 tissue culture cells.

Members of the L1 family of homophilic neural cell adhesion molecules are thought to play an important role in nervous system development and function. It is also suggested that L1 is a direct target of ethanol in fetal alcohol syndrome, since ethanol inhibits the aggregation of cultured cells expressing L1 (Ramanathan, R., Wilkemeyer, M. F., Mittel, B., Perides, G., and Charness, M. E. (1996) J. Cell Biol. 133, 381-390). If ethanol acts directly on the homophilic adhesive function of the L1 molecule, then inhibition of aggregation by ethanol should be observed in any cell type that expresses L1. Here we examined the effect of physiologically relevant concentrations of ethanol on the aggregation of Drosophila S2 cells that expressed either neuroglian (the Drosophila homolog of L1) or human L1. The aggregation of these S2 cells is known to be solely dependent on the homophilic interactions between L1 or neuroglian molecules. Neither cell adhesion molecule was affected when cell aggregation assays were carried out in the presence of >/=38 mM ethanol. The recruitment of membrane skeleton assembly at sites of cell-cell contact (a transmembrane signaling function of human L1) was also unaffected by the presence of ethanol. Thus the previously described inhibition of cell adhesion by ethanol in L1-expressing cells cannot be explained by a simple direct effect on the adhesive activity of L1 family members.