A serotonergic system in the brain of the Antarctic fish, Trematomus bernacchii

The immunohistochemical distribution of serotonin-containing nerve fibres and cells has been described in the brain of the Antarctic fish, Trematomus bernacchii. The largest serotonergic system was associated with the diencephalic and rhombencephalic ventricles. In particular, serotonin-positive cells have been found in the lateral recess and neuropile zone of the diencephalic ventricle, where we have identified the serotonergic portion of the paraventricular organ. Numerous serotonin cells were localized in the dorsal nucleus of the raphe, the dorsal tegmental nucleus and the central gray. Two large cell groups, arranged in a pair of well-defined columns and connecting the central gray with the dorsal reticular formation, were immunostained in the region of the trigeminal nuclei. In addition, few positive cells have been found in the preoptic area and the cerebellar valvula, and few serotonergic nerve fibres, probably belonging to the lateral lemniscus, have been identified. The distribution of serotonin elements in the brain of T. bernacchii has been compared with that described in other fish, where it showed some modifications in the immunoreactive pattern. Finally, the lack of a serotonergic system at the level of the reticular superior formation has been reported; however, it was not possible to rule out a phylogenetic or environmental explanation.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

Gas exchange studies in two Portuguese grapevine cultivars

Gas exchange characteristics of leaves of Vitis vinifera L. cvs Tinta Amarela and Periquita, two grapevine cultivars grown in distinct climatic regions of Portugal, were studied under natural and controlled conditions. Daily time courses of gas exchange were measured on both a hot, sunny day and a cooler, partly cloudy day. Responses of net photosynthesis to irradiance and internal partial pressure of CO₂, were also obtained. A strong correlation between net photosynthesis (P_N) and leaf conductance (g_s) was found during the diurnal time courses of gas exchange, as well as a relatively constant internal partial pressure of CO₂ (P_i), even under non-steady-state conditions. On the cloudless day, both P_N and g_s were lower in the afternoon than in the morning, despite similar conditions of leaf temperature, air to leaf water vapor deficit and irradiance. The response curves of net photosynthesis to internal CO₂ showed linearity up to p_i values of 50 Pa, possibly indicating a substantial excess of photosynthetic capacity. When measured at low partial pressures of O₂ (1 kPa), P_N became inhibited at high CO₂ levels. Inhibition of P_N at high CO₂ was absent under normal levels of O₂ (21 kPa). Significant differences in gas exchange characteristics were found between the two cultivars, with T. Amarela having higher rates under similar measurement conditions. In particular, the superior performance of T. Amarela at high temperatures may represent adaptation to the warmer conditions at its place of origin.     

Transport of l(-)malic acid and other dicarboxylic acids in the yeast Candida sphaerica

Summary dl-Malic acid grown cells of Candida sphaerica (anamorph of Kluyveromyces marxianus) formed a saturable transport system that mediated accumulative transport of l(-)malic acid with the following kinetic parameters at pH 5.0: V _max, 0.44 nmol l(-)malate·s^-1 per milligram dry weight; K _m ,0.1 mM l(-)malate. Initial uptake of the acid was accompanied by disappearance of extracellular protons, the rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. Variation with extracellular pH of the K _m values, calculated either as the concentrations of anions or of undissociated acid, pointed to anions as the transported form. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonylcyanide-M-chlorophenyl-hydrazone (CCCP). These results suggested that the transport system was a dicarboxylate-proton symporter. The system was inducible and was subject to glucose repression. Succinic, fumaric, -ketoglutaric, oxaloacetic and d-malic acid, but not maleic, malonic, oxalic nor l(+)-tartaric acid, apparently used the same transport system since they acted as competitive inhibitors of l(-)malic acid transport and induced proton movements that followed Michaelis-Menten kinetics. Experiments with glucose-repressed cells showed that undissociated dicarboxylic acid (measured with labelled succinic acid) entered the cells slowly by simple diffusion. The permeability of the cells for undissociated acid increased exponentially with pH, the diffusion constant increasing 100-fold between pH 3.5 and 6.0.     

Differential binding of anions to the active site of Cu,Zn superoxide dismutase. A study of the Co,Zn enzyme derivative

The reaction of N3- with Co,Zn superoxide dismutase, a good analogue of the native Cu,Zn enzyme, was studied in the presence and absence of phosphate, which is known to perturb the spectroscopic properties of the cobalt chromophore in the Co,Zn enzyme. EPR, NMR, and optical titrations demonstrated the formation of different adducts for N3- depending on the presence of phosphate, at variance with results previously obtained with CN- [3]. This evidence indicates that the mechanism of anion binding to Cu,Zn superoxide dismutase cannot be described on the basis of data obtained with a single type of anions.     

Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin.

1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.     

Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalemic periodic paralysis on chromosome 17

Summary Paramyotonia congenita (PC), an autosomal dominant non-progressive muscle disorder, is characterised by cold-induced stiffness followed by muscle weakness. The weakness is caused by a dysfunction of the sodium channel in muscle fibre. Parts of the gene coding for the -subunit of the sodium channel of the adult human skeletal muscle (SCN4A) have been localised on chromosome 17. To investigate the role of this gene in the etiology of PC, a linkage analysis in 17 well-defined families was carried out. The results (z=20.61, =0.001) show that the mutant gene responsible for the disorder is indeed tightly linked to the SCN4A gene. The mutation causing hyperkalemic periodic paralysis (HyperPP) with myotonia has previously been mapped to this gene locus by the same candidate gene approach. Thus, our data suggest that PC and HyperPP are caused by allelic mutations at a single locus on chromosome 17.Dedicated to Professor P. E. Becker on the occasion of his 83rd birthday.     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin.

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.