Características morfológicas de la retina del calderón común (Globicephala melas; traill, 1809) y su relación con el hábitat / Morphological characteristics of pilot whales retina (Globicephala melas; traill, 1809) and their relationship to habitat

Existe una estrecha relación entre las características morfológicas de los sistemas sensoriales, su funcionamiento y el hábitat al que están adaptados los organismos. En este sentido, de todos los mamíferos marinos estudiados, los cetáceos son los que más profundamente han modificado su estructura y fisiología ocular por su estrecha adaptación a una vida exclusivamente acuática. Para aportar más datos a la literatura, el objetivo de este trabajo es describir morfológicamente la retina de la ballena piloto a través de técnicas de microscopia óptica, con el fin de relacionarla con su adaptación al medio acuático. Nuestros datos muestran que la retina de Globicephala melas se organiza de acuerdo al mismo plan básico de los vertebrados. Tiene un grosor medio alrededor de 330±23 µm en las zonas de alta densidad de células ganglionares y 175±2 µm en la zona periférica. La capa de los fotorreceptores se corresponde con el 45% del grosor de la retina total. Presenta largos segmentos externos. La capa más característica de cetáceos en general y de Globicephala melas en particular, es la capa de células ganglionares. Su grosor, de 77,76±37,26 siendo la más variable de toda la retina. Esta capa presenta baja densidad celular pero tamaños excepcionalmente grandes, de 10 a 75 µm (promedio de 33,5 µm), denominadas células ganglionares gigantes.

There is a close relationship between morphological features of sensory systems, their function and habitat to which these organisms are adapting. In this sense, of all marine mammals that have been studied, cetaceans are the ones that have profoundly changed structure and ocular physiology in their adaptation to an exclusively aquatic life. To add further data to the literature, the aim of this paper is to describe morphologically the retina of the pilot whale through optical microscopy and relate their adaptation to the aquatic environment. Our data show that the retina of the long-finned pilot whale is organized according to the same basic plan of vertebrates. It has an average thickness of about 330±23 microns in areas of high ganglion cell density of 175±2 microns in the peripheral zone. Photoreceptor layer corresponds to 45% of total thickness of the retina and has long outer segments. The most significant characteristic of cetaceans in general and long-finned pilot whale in particular, is the ganglion cell layer. Thickness of 77.76±37.26 being the most variable of the entire retina. This layer has a low density but exceptionally large cell size of 10 to 75 microns (average of 33.5 microns), known as giant ganglion cells.

[Thrombophilic factors in divers with undeserved decompression sickness]. / Bilan de thrombophilie chez des plongeurs après un accident de décompression immérité.

UNLABELLED: In divers with a vascular disease in decompression sickness, who have not committed any technical error, thrombophilic risk factors were sought. Six cases of confirmed divers, without diving technical error, were investigated. Thrombophilic screening included proteins C, S, antithrombin III, and factor VIII assays, and circulating antibodies, Factor V Leiden, and mutation G20210A mutation in Factor II gene research. Total plasma homocysteine (Hcy), an atherosclerosis factor, even when slightly increased, nutitional factors: folate and vitamins B12 and B6, the cofactors of its metabolism, and inversely correlated with Hcy values, were assayed, and subjects were genotyped for mutation C677T in the MTHFR gene. RESULTS: In five divers, Hcy values were moderately increased, and in all the six, folate and/or B12 values were decreased. Three of them showed a genotype TT (mutation C677T), two, the genotype CT, and the sixth, an heterozygous Factor V Leiden. In these divers, a predisposition for vascular diseases, was detected, which was partially curable.

Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome.

The voltage-dependent K(+) channel responsible for the slowly activating delayed K(+) current I(Ks) is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K(+) and Na(+) intakes. On a normal K(+) diet, homozygous kcne1(-/-) mice exhibit signs of chronic volume depletion associated with fecal Na(+) and K(+) wasting and have lower plasma K(+) concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K(+) diets or stimulated by low Na(+) diets, a high K(+) diet provokes a tremendous increase of plasma aldosterone levels in kcne1(-/-) mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K(+) in kcne1(-/-) mice. This exacerbated aldosterone production in kcne1(-/-) mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I(Ks) may directly participate in the control of aldosterone production by plasma K(+). These results, which show that KCNE1 and I(Ks) are involved in K(+) homeostasis, might have important implications for patients with I(Ks)-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Relations between the stage of cell maturation and lactate transporter activities in rat neonatal muscle cells in culture.

Lactate transport was investigated in newborn rat muscle cells in culture. The aim was to study the lactate transport function at two stages of cell differentiation in culture: (i) during the proliferative phase characterized by myoblasts and myotubes (MyB/MyT2) obtained after 2-3 seedings, (ii) when myotubes (MyT1) grow old in culture after 8-9 seedings. In both developmental stages MyB/MyT2, lactate was carried following a saturable and sigmoidal velocity curve: the Hill and the Scatchard plot analyses confirmed an allosteric or multisite mechanism of lactate transport with two classes of carriers: one of low and one of high affinity i.e., 8.6 and 0.95 mm, respectively, which are associated with high and low transport capacities (V(m)) i.e., 9.1 and 0.67 nm/min/mg, respectively. With MyT1, the velocity curve of lactate transport presented a hyperbolic profile, and the Hill plot analysis gave a Hill number near one suggesting that for cell aging in culture the decrease in cooperativity shows that lactate transport essentially occurs through the low affinity transport system. Inhibitor effects also contributed to evidence for at least two systems of transport. Results obtained from primary cells give evidence for the early activity of lactate transport system at the Myb/MyT2 stage and its evolution during cell aging in culture (MyT1). Sarcolemmal lactate transport in primary cultures of myocytes is accomplished by multiple carriers, neither of which are MCT1 or MCT2 as confirmed by immunoblots.

Genetic analysis of the beta subunit of the epithelial Na+ channel in essential hypertension.

Mutations of the last exon of the beta subunit of the amiloride-sensitive epithelial Na+ channel (betaENaC) can lead to Liddle's syndrome, a rare monogenic form of hypertension. The objective of this study was to test whether more subtle changes of betaENaC could be implicated in essential hypertension. After determination of the betaENaC coding gene organization (12 exons spanning 23.5 kb), a systematic screening of the last exon of the gene was performed in 525 subjects (475 whites, 50 Afro-Caribbeans), all probands of hypertensive families. This search was extended to the remaining 11 exons in a subset of 101 probands with low-renin hypertension. Seven amino acid changes were detected: G589S, T594M, R597H, R624C, E632G (last exon), G442V, and V434M (exon 8). These genetic variants were more frequent in subjects of African origin (44%) than in whites (1%). The functional properties of the variants were analyzed in Xenopus oocytes by two independent techniques, ie, electrophysiology and 22Na+ uptake. Small but not significant differences were observed between the variants and wild type. The clinical evaluation of the family bearing the G589S variant, which provided the highest relative ENaC activity, did not show a cosegregation between the mutation and hypertension. The present study illustrates the difficulty in establishing a relation of causality between a susceptibility gene and hypertension. Furthermore, it does not favor a substantial role of the betaENaC gene in essential hypertension.

Lactate transport in L6 skeletal muscle cells and vesicles: allosteric or multisite mechanism and functional membrane marker of differentiation.

Membrane lactate transport was studied in skeletal muscle cells and membrane vesicles from the L6 line in relation to in vitro myogenesis. In myoblasts, lactate was transported by simple diffusion and insensitive to classical inhibitors: a positive correlation between onset of creatine kinase activity and lactate transport in differentiated myotubes was observed and could be considered to be a functional marker of cell differentiation. In myotubes, complete analysis of the velocity curves (direct coordinates, Eadie-Scatchard plots, Hill plots) gave parameters showing that lactate was carried by an allosteric or multisite system. This was confirmed by using sarcolemmal vesicles and specific inhibitors. In whole cells, alpha-cyano-4-hydroxycinnamic acid (CIN) and parachloromercuribenzylsulphonic acid (pCMBS) inhibited the maximal velocity without modifying the global cooperativity of the system. The weak effect of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), which has a low affinity constant (Ki = 22.5 microM), implicated the monocarboxylate system rather than the anionic exchanger as a carrier system in muscle cells. CIN and DIDS exhibited one type of interaction with lactate carriers, and the curvilinear shape of the lactate Hill plot with or without inhibitors suggested that inhibitors were active at the same family of interaction sites and had a common range of affinities. The apparent competitive inhibition of pyruvate (Ki = 3.2 mM) did not modify the transport pathway of lactate in L6 myotubes. In conclusion, kinetic analysis of lactate transport in the presence or absence of inhibitors gave evidence for a multisite lactate carrier activity in myotubes composed of two systems at least, related to two or three isoforms of lactate carriers.

Kinetic asymmetry of renal Na+-L-lactate cotransport. Characteristic parameters and evidence for a ping pong mechanism of the trans-stimulating exchange by pyruvate.

Brush border vesicles prepared from horse renal cortex were used to study the kinetic properties of the Na+-L-lactate carrier on the outer and inner faces of the membrane. Two methods were applied for these measurements (in the absence of an electrical gradient): a direct method using influx and efflux kinetics, and an indirect method applied to trans-stimulated influx kinetics using membrane vesicles preloaded with various pyruvate concentrations (the latter enabled us to observe simultaneously the inner and outer carrier properties). Kinetic parameters obtained by the first method have shown that under sodium lactate chemical gradient, the carrier efficiency (estimated by the ratio of k = Vm/Km) is higher for the influx than efflux, a mechanism indicating a kinetic asymmetry of the transport. This difference remains at chemical equilibrium of solute concentration. The similarity of outer and inner affinity of sodium permits one to conclude that the kinetic asymmetry of the sodium lactate transport is related to the lactate-carrier interaction and not to that of the sodium-carrier. The second method using the pyruvate trans-activation effect (under sodium chemical equilibrium) has shown an affinity of lactate (Kt(out) = 1.1 mM), about 15 times higher for the carrier in the extracellular orientation than that of pyruvate for the carrier in the intracellular orientation (Kt(pyr) = 36 mM). This method has demonstrated a ping pong mechanism for the trans-activation exchange which accounts for a selective pore carrier model like a gated channel. These asymmetric properties are related to the AS glide sequential model (A and S being Na+ and lactate, respectively) proposed previously for the Na-L-lactate cotransport and to a different accessibility of the organic solute but not of the sodium on the two membrane faces.

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles.

The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zero trans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and delta psi = 0. Under zero trans condition and delta psi = 0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (Kt = 88 microM) and a low affinity for sodium (Kt = 57.7 mM) (site I), the second one with a low affinity for pyruvate (Kt = 6.1 mM) and a high affinity for sodium (Kt = 23.9 mM) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25 mM and 8 mM pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously. Under chemical equilibrium (delta psi congruent to 0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation was n = 1.7. The direct method of measuring Na+/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives a n = 1.67. Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).

LLC-PK1 cells express Na+-lactate cotransport in apical membranes after confluency.

L-[3H]lactate uptake was characterized in LLC-PK1 cell apical membrane vesicles obtained by intensive culture on microcarrier beads. The apical membrane preparation technique involved MgCl2 precipitation. Na+-dependent L-[3H]lactate uptake was present only after confluency; its appearance paralleled the subcellular localization of aminopeptidase in apical membranes. L-[3H]lactate uptake was Na+-dependent and electrogenic. Only the Na+-dependent component of L-[3H]lactate uptake was saturable with one family of independent carriers. The apparent affinity constant was 1.1 +/- 0.25 mM and the apparent maximal velocity was 29 +/- 3 nmol.mg-1.min-1. The Na+-lactate cotransport stoichiometry was 2 Na+ for 1 lactate. The specificity of the L-lactate transport system was compatible with that of the monocarboxylic acid pathway described previously in brush-border membranes of kidney cortex and discrete from the tricarboxylic acid carrier, the D-glucose transporter, and the general pathway for anions. The LLC-PK1 cell line appears to be a useful tool for study of the regulation of L-lactate uptake and biosynthesis of the renal monocarboxylic acid transporter.

The mechanism of Na+-L-lactate cotransport by brush-border membrane vesicles from horse kidney. Analysis by isotopic exchange kinetics of a sequential model and stoichiometry.

The present study determines the characteristics of isotopic Na and lactate exchange under equilibrium conditions in horse kidney brush-border membrane vesicles. The influence of one solute (Na+ or lactate) on the isotopic exchange of the co-transported species (lactate or Na) was analyzed in detail. Analysis of the data suggests that Na and lactate interact sequentially with the carrier. The observed apparent symmetry between the activating effect of low Na concentrations and the inhibiting effect of high Na concentrations on the lactate exchange process suggests that the carrier functions according to a glide symmetry model where the two solutes bind to the carrier according to an iso-ordered Bi Bi process. This conclusion is strengthened by the properties of the 22Na exchange process catalyzed by the lactate carrier. Direct measurement under equilibrium conditions of the coupled Na+/lactate transport indicated a stoichiometry of 2:1. These data, along with those obtained in a previous study (Mengual R., and Sudaka P. (1983) J. Membr. Biol. 71, 163-171) allow us to propose a mechanism for the Na+-lactate cotransport, involving a transport system that functions according to an iso-ordered Bi Bi process where binding and debinding of Na+ occur first, followed by the lactate molecule.

The mechanism of Na+-L-lactate cotransport by brush border membrane vesicles from horse kidney: analysis of rapid equilibrium kinetics in absence of membrane potential.

Membrane transport of lactate was studied using vesicles prepared from horse kidney brush border. It is shown that the carrier-mediated transport of L-lactate is Na dependent and the D-lactate Na dependence seems weaker than the L stereoisomer. Augmented transport rate is observed following imposition of an artificial chemical Na+ gradient of electrical potential difference. The effect of Na+ chemical gradient on the L-lactate uptake was analyzed using membrane vesicles incubated with 50 mM KCl and valinomycin in order to short circuit any contribution of transmembrane electrical potential to the transport. Kinetics results and principally the absence of linearity between l/v (lactate) versus l/Na+ show that the L-lactate transport mechanism fit the properties of an ordered process with two Na+ ions cotransported with one L-lactate anion. The L-lactate and sodium affinities (Km) determined under Na+ chemical gradient were 1.05 and 48 mM for L-lactate and Na, respectively. The sodium activation was shown to be highly cooperative with a Hill number of 2 although no "sigmoidal" activation effect was observed.

Large-scale purification of the 3'-OH-terminal tRNA-like sequence (n = 159) of turnip-yellow-mosaic-virus RNA.

In order to undertake structural and functional studies on the 3'-terminal part of turnip yellow mosaic virus RNA, a structure which can be specifically aminoacylated by valyl-tRNA synthetase, we have developed large-scale methods for purifying the tRNA-like sequence. Several experimental approaches were tested. One procedure was retained enabling us to purify large quantities of the homogeneous tRNA-like fragment. Starting from 1.5 g turnip yellow mosaic virus, one obtains 400 mg RNA, which is partially digested by T1 ribonuclease and which yields 1-2 mg pure tRNA-like fragment after three chromatographic steps: two filtrations on Ultrogel ACA 54 and one reverse-phase chromatography (RPC 5) in the presence of urea. A method has been worked out allowing preparation of 10 mg of the fragment per month. The purified RNA material appeared homogeneous upon polyacrylamide gel electrophoresis under denaturing conditions. The isolated tRNA-like structure can be valylated to an extent of 100% in the presence of purified yeast valyl-tRNA synthetase with kinetic parameters resembling those of the tRNAVal aminoacylation.

Valylation of the two RNA components of turnip-yellow mosaic virus and specificity of the tRNA aminoacylation reaction.

A comparative study of the aminoacylation of the two RNA components of turnip yellow mosaic virus, of yeast tRNAVal, tRNAfMet and of tRNAPhe by purified yeast valyl-tRNA synthetase is reported. Aminoacylations were performed in the presence of pure yeast tRNA nucleotidyltransferase, since 85% of the viral RNA molecules lacked the 3'-adenosine. We find that aminoacylation of the viral RNAs, like tRNA aminoacylation, reflects an equilibrium between the acylation and deacylation reactions. The kinetic parameters of TYM virus RNA valylation resemble the values found for tRNAVal valylation; in particular, there is a strong affinity between the viral RNA and valyl-tRNA synthetase and the rate constant for TYM virus RNA valylation is only slightly lower than that for tRNAVal. This result contrasts with the reduced rates observed in tRNA mischarging, and suggests that the viral RNA could be easily aminoacylated in vivo. Considering the fact that the 3'-terminal sequence of TYM virus RNA has only a few points of resemblance to a tRNA sequence, we propose that there are some structural motifs found in both tRNAVal and TYM virus RNA which are brought in a similar spatial arrangement recognized by valyl-tRNA synthetase.