Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

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.     

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor,peripheral ν-polypeptide (Mr 43,000)

NaIO₄ oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (α, β, γ and δ) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ν-peptide (M_r 43,000), pH and temperature. Very low NaIO₄ concentrations (50 μM) suffice to prevent the penetration of the ν-peptide into NaDodSO₄ polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (α, M_r 40,000) into a doublet. In contrast, NaIO₄ treatment of the detergent-solubilized receptor aggregated the α-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO₄-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (～2 mM NaIO₄).Neuraminidase digestion affected exclusively the γ- and δ-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. β-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO₄ nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-α-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and β-glucosidase treatments. The latter enzymatic digestion reduced the α-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.     

Myotonia congenita (Thomsen's disease) excluded from the region of the myotonic dystrophy locus on chromosome 19

Summary Linkage analysis has been carried out in six German families with autosomal dominantly inherited myotonia congenita (Thomsen's disease) using five chromosome 19 markers known to be linked to the gene for myotonic dystrophy (DM). Two of the markers, APOC1 and APOC2, are tightly linked to DM. Close linkage between these markers and myotonia congenita (MC) has been excluded to a distance of 9cM (z=-2.158). These data support the clinical suggestion that MC and DM are non-allelic disorders.     

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.     

Developmental studies on creatine kinase. Isoenzyme in rat gastrointestinal tract

Creatine kinase activity and its isoenzymatic profile in rat intestinal mucose during normal development have been studied. Creatine kinase enzymatic activity increased stepwise during fetal development and the first week of life. An isoenzymatic pattern of exclusively CK-BB types occurred in all segments of the digestive tract during the early fetal stage. The isoenzyme profile of creatine kinase in the esophagic tissue with advancing maturation of the fetus shifted in the same way as in adults, with preferential concentration of CK-MM. However, CK-BB continued to be the main isoenzyme in the rest of the digestive tract. Our results show that rats are particularly suitable for experimental studies of intestinal creatine kinase isoenzymes.     

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.     

Influence of blood proteins in the in vitro adhesion of Staphylococcus epidermidis to teflon, polycarbonate, polyethylene and bovine pericardium.

The influence of human plasma proteins (fibrinogen, albumin and fibronectin) on the adherence of Staphylococcus epidermis to teflon, polyethylene, polycarbonate and bovine pericardium was studied in an in vitro quantitative assay by scintillation counting. Bacterial adhesion was generally reduced by the presence of protein during the adherence assay except in the case of bovine pericardium, in which adherence remained almost unaffected. The effect of these plasma proteins on bacterial surface properties resulted in strong increases of surface charge as measured by ion-exchange chromatography and with no effect on hydrophobicity, estimated as contact angles. Adherence was not found to be correlated with these two properties, suggesting that bacteria-surface interactions must not be simplified to the influence of interfacial forces.     

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.