Sera from amyotrophic lateral sclerosis patients induce the non-canonical activation of NMDA receptors "in vitro".

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR. Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique. Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP(3) receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects. Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.

Factors involved in alleviating water stress by partial crop removal in pear trees.

We studied the relief of water stress associated with fruit thinning in pear (Pyrus communis L.) trees during drought to determine what mechanisms, other than stomatal adjustment, were involved. Combinations of control irrigation (equal to crop water use less effective rainfall) and deficit irrigation (equal to 20% of control irrigation), fruit load (unthinned and thinned to 40 fruits per tree) and root pruning (pruned and unpruned) treatments were applied to pear (cv. 'Conference') trees during Stage II of fruit development. Daily patterns of midday stem water potential (Psi(stem)) and leaf conductance to water vapor (g(l)) of deficit-irrigated trees differed after fruit thinning. In response to fruit thinning, gl progressively declined with water stress until 30 days after fruit thinning and then leveled off, whereas the effects of decreased fruit load on Psi(stem) peaked 30-40 days after fruit thinning and then tended to decline. Soil water depletion was significantly correlated with fruit load during drought. Our results indicate that stomatal adjustment and the resulting soil water conservation were the factors determining the Psi(stem) response to fruit thinning. However, these factors could not explain differences in daily patterns between g(l) and Psi(stem) after fruit thinning. In all cases, effects of root pruning treatments on Psi(stem) in deficit-irrigated trees were transitory (Psi(stem) recovered from root pruning in less than 30 days), but the recovery of Psi(stem) after root pruning was faster in trees with low fruit loads. This behavior is compatible with the concept that the water balance (reflected by Psi(stem) values) was better in trees with low fruit loads compared with unthinned trees, perhaps because more carbon was available for root growth. Thus, a root growth component is hypothesized as a mechanism to explain the bimodal Psi(stem) response to fruit thinning during drought.

Response of winter root starch concentration to severe water stress and fruit load and its subsequent effects on early peach fruit development.

Effect of water stress during stage III of peach fruit development on winter root starch concentration (RSC) and subsequent reproductive development was studied. Two irrigation treatments were applied in two consecutive seasons (2003-2004): full irrigation (FI) and no irrigation during stage III of fruit development until visible leaf wilting (LWI), which occurred when midday stem water potential reached -1.80 MPa. Three fruit thinning intensities were applied within each irrigation treatment. The year 2005 was a recovery year in which all trees received full irrigation and commercial fruit thinning. Water deficit and high fruit loads in the previous season significantly reduced the concentration of winter RSC. Fruit set and fruit growth from full bloom to 30 days after full bloom (30 DAFB) increased with increasing winter RSC before other factors, such as inter-fruit competition and availability of carbon from current photosynthesis, came into play. Consequently, severe water stress reduced the total number of fruits and fruit dry mass growth 30 DAFB. However, during the recovery year and after fruit thinning, fruit loads were similar between irrigation treatments and yield capacity remained unaffected. Peach fruit production recovered quickly from the deleterious effects of two consecutive years of water stress because of a combination of two factors: (1) reduced initial fruit set that was still adequate to achieve a commercial crop; and (2) the low sensitivity of fruit growth 30 DAFB to winter RSC.

Growth patterns and morphology of fine roots of size-controlling and invigorating peach rootstocks.

We compared growth patterns and morphology of fine roots of size-controlling and invigorating peach (Prunus persica (L.) Batsch) rootstocks. Peach trees were grafted on five rootstocks: a vigorous control (Nemaguard), three intermediate vigor rootstocks (K119-50, P30-135 and Hiawatha), and a semi-dwarfing rootstock (K146-43). Minirhizotron tubes were installed at the base of trees on each rootstock and root images captured with a minirhizotron digital camera system. Number, visible length, and diameter of new roots were recorded at fixed soil depths from April 19, 2000 to December 19, 2001. Root diameter, specific root length, root tissue density and root length density were also measured periodically for each rootstock on roots collected from in-growth cores. Rootstocks had similar seasonal patterns of new root production. Fine root production was lowest in winter and appeared to decline during the final stages of fruit growth. A rootstock with almond in its genetic background (K119-50) produced the greatest quantity of fine roots and had the greatest number of new roots below 69 cm, whereas there were no differences among the other four rootstocks in the total number of roots produced. Rootstock K146-43 had thicker fine roots than the other rootstocks. Independent of rootstock, fine roots produced during spring had greater specific root length than those produced later in the season. The seasonal pattern of fine root production did not appear to be associated with the previously reported effects of these dwarfing rootstocks on shoot growth and stem water potential early in the growing season.

Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ.

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.

Mitigation of effects of extreme drought during stage III of peach fruit development by summer pruning and fruit thinning.

A water deficit during stage III of fruit growth was established with the aim of determining if it is possible to achieve an improvement in tree water status by summer pruning and fruit thinning. The experiment was set up as a randomized block split-plot design across trials (irrigation) where pruning was assigned to the main plot and fruit thinning to the sub-plots. The irrigation treatments were (1) standard full irrigation (FI), and (2) suppression of irrigation during stage III of fruit growth until leaves visibly withered (LWI); the pruning treatments were (1) experimental summer pruning (EP), and (2) standard summer pruning (CP); and three fruit thinning intensities were applied to facilitate analysis of the effects of the treatments in relation to fruit load. Changes in amount of light intercepted and in tree stem water potential (Psi stem) were evaluated. The EP treatment reduced the amount of light intercepted by the tree. In the FI treatment, there was a significant reduction in fruit growth measured as both water accumulation and dry mass accumulation. Under FI conditions, reductions in fruit load as a result of EP were not accompanied by a significant improvement in Psi stem. In the LWI treatment, EP produced a significant improvement of 0.17 MPa in Psi stem, but there was no improvement in fruit growth compared with CP trees. A reduction in fruit load from 350 (commercial load) to 150 per tree significantly improved Psi stem by 0.3 MPa at the end of stage III of fruit growth. These results indicate that improvements in water status in response to pruning may be insufficient to promote fruit growth if the pruned trees are unable to provide an adequate supply of assimilates to the developing fruits.

Endogenous hemichannels play a role in the release of ATP from Xenopus oocytes.

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium-free solution and inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium-sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes.

Cholinergic currents in Xenopus oocytes transplanted with human brain membranes

Corrientes colinérgicas de cerebro humano fueron registradas en oocitos de Xenopus laevis trasplantados con membranas de cerebro humano procedentes de dos zonas diferentes, la corteza frontal y el hipocampo. Las corrientes registradas fueron activadas por el receptor nicotínico o por el receptor nicotínico o muscarínico de la acetilcolina. Se probaron los efectos de diferentes agonistas nicotínicos como acetilcolina, nicotina y yoduro de 1,1-dimetil-4-fenil-piperazinio (DMPP), y antagonistas del receptor nicotínico como a-bungarotoxina y d-tubocurarina en los oocitos transplantados. Detectamos cuatro clases de cinéticas de corrientes nicotínicas. Las diferencias en la amplitud y en la carga eléctrica total de las corrientes provocadas por varios agonistas en el rango de potencial mantenido no fueron significativas, excepto en el caso del DMPP a un potencial mantenido de -90 mV. Nuestros resultados indican que las formas alfa4beta2, alfa3beta4 y alfa7 son los principales receptores nicotínicos en el cerebro humano

Cholinergic human brain currents were recorded in Xenopus laevis oocytes transplanted with human cerebral membranes from two different zones, the frontal cortex and the hippocampus. The recorded currents were supported by the nicotinic or the muscarinic acetylcholine receptor. We tested the effects of a number of several nicotinic agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and the nicotinic receptor antagonists a-bungarotoxin and d-tubocurarine on the transplanted oocytes. We detected four kinds of nicotinic current kinetics. The differences in the amplitude and in the total electric charge of the currents elicited by various agonists at a range of holding potentials were not significant, except in the case of DMPP at a holding potential of -90 mV. Our results indicate that alpha4beta2, alpha3beta4 and alpha7 are the main nicotinic receptors in human brain

Effect of galantamine on the human alpha7 neuronal nicotinic acetylcholine receptor, the Torpedo nicotinic acetylcholine receptor and spontaneous cholinergic synaptic activity.

1. Various types of anticholinesterasic agents have been used to improve the daily activities of Alzheimer's disease patients. It was recently demonstrated that Galantamine, described as a molecule with anticholinesterasic properties, is also an allosteric enhancer of human alpha4beta2 neuronal nicotinic receptor activity. We explored its effect on the human alpha7 neuronal nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. 2. Galantamine, at a concentration of 0.1 microM, increased the amplitude of acetylcholine (ACh)-induced ion currents in the human alpha7 nAChR expressed in Xenopus oocytes, but caused inhibition at higher concentrations. The maximum effect of galantamine, an increase of 22% in the amplitude of ACh-induced currents, was observed at a concentration of 250 microM Ach. 3. The same enhancing effect was obtained in oocytes transplanted with Torpedo nicotinic acetylcholine receptor (AChR) isolated from the electric organ, but in this case the optimal concentration of galantamine was 1 microM. In this case, the maximum effect of galantamine, an increase of 35% in the amplitude of ACh-induced currents, occurred at a concentration of 50 microM ACh. 4. Galantamine affects not only the activity of post-synaptic receptors but also the activity of nerve terminals. At a concentration of 1 microM, quantal spontaneous events, recorded in a cholinergic synapse, increased their amplitude, an effect which was independent of the anticholinesterasic activity associated with this compound. The anticholinesterasic effect was recorded in preparations treated with a galantamine concentration of 10 microM. 5. In conclusion, our results show that galantamine enhances human alpha7 neuronal nicotinic ACh receptor activity. It also enhances muscular AChRs and the size of spontaneous cholinergic synaptic events. However, only a very narrow range of galantamine concentrations can be used for enhancing effects.

Kainate-triggered currents in Xenopus oocytes injected with chick retinal membrane fragments: effect of guanine nucleotides.

PURPOSE: To electrophysiologically characterize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection. METHODS: A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at -70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining. RESULTS: Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% +/- 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 +/- 72.3 microm(2)). Bath-applied kainate induced dose-dependent (EC(50): 64 +/- 7 microM), nondesensitizing inward currents (15-90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by gamma-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner. CONCLUSIONS: An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.

Daily shoot extension growth of peach trees growing on rootstocks that reduce scion growth is related to daily dynamics of stem water potential.

We studied relationships between diurnal patterns of stem water potential (PsiSTEM) and stem extension growth of the same scion cultivar growing on three rootstocks with differing size-controlling potentials. The peach trees (Prunus persica (L.) Batsch) used in this field experiment consisted of an early-maturing freestone cultivar, 'Flavorcrest,' grafted onto three different rootstocks: Nemaguard (a vigorous seed-propagated control, P. persica x P. davidiana hybrid), Hiawatha (an intermediate vigor rootstock, derived from an open pollinated seedling of a P. besseyi x P. salicina hybrid) and K-146-43 (a semi-dwarfing rootstock, P. salicina x P. persica hybrid). Diurnal patterns of PsiSTEM and stem extension growth were measured on six dates (March 29, April 12, April 26, May 10, May 24 and June 18) during the primary period of peach shoot extension growth. Rootstocks clearly affected diurnal patterns of PsiSTEM and stem extension growth. Trees on K-146-43 had the lowest midday PsiSTEM and stem extension growth. Differences among rootstocks in the amount of diurnal oscillation in PsiSTEM explained stem extension rate differences induced by the three rootstocks. The sensitivity of shoot extension growth to tree water relations tended to decrease as the season progressed and was not apparent by mid-June. The results of the study indicate that water relations may play an important role in the dwarfing mechanism induced by size-controlling peach rootstocks.

Influence of branch autonomy on fruit, scaffold, trunk and root growth during stage III of peach fruit development.

We studied the influence of branch autonomy on the growth of reproductive and vegetative organs by establishing different patterns of fruit distribution within and between large branch units (scaffolds) in mature peach trees (Prunus persica (L.) Batsch cv. 'Elegant Lady'). Different patterns of fruit distribution were established by defruiting either whole scaffolds (uneven fruit distribution between scaffolds; US) or several selected hangers (small fruiting branches) per tree (uneven fruit distribution between hangers; UH). The effects of these patterns were compared with the effects of an even fruit distribution treatment (EVEN) in which fruits were thinned to achieve maximum uniformity of fruit distribution within the canopy. The desired fruit loads were obtained by differentially thinning the remaining bearing parts. On a tree basis, the response of mean fruit mass to fruit load was strongly affected by fruit distribution. The steepest mean fruit mass to fruit load relationship was found in US trees, whereas the relationship in UH trees was intermediate between the US and EVEN trees. On a scaffold basis, differences in fruit size between EVEN and US trees with similar fruit loads, though statistically significant, were relatively small, indicating that scaffolds were almost totally autonomous with respect to dry matter partitioning to fruit during the final stage of peach fruit growth. Hangers also appeared to exhibit significant autonomy with respect to the distribution of dry matter during the final phase of fruit growth. Branch autonomy was evident in scaffold growth: defruited scaffolds in the US treatment grew more than fruited scaffolds, and fruit distribution treatments had little impact on scaffold cross-sectional area on a tree basis. On the other hand, as observed for fruit growth, branch autonomy did not appear to be complete because the fruited scaffolds grew more in US trees than in EVEN trees under heavy cropping conditions. However, the effect of fruit distribution occurred only over short distances, and was negligible on organs located farther away from the source of heterogeneity (fruits), such as the trunk and roots.

Control of neurotransmitter release by an internal gel matrix in synaptic vesicles.

Neurotransmitters are stored in synaptic vesicles, where they have been assumed to be in free solution. Here we report that in Torpedo synaptic vesicles, only 5% of the total acetylcholine (ACh) or ATP content is free, and that the rest is adsorbed to an intravesicular proteoglycan matrix. This matrix, which controls ACh and ATP release by an ion-exchange mechanism, behaves like a smart gel. That is, it releases neurotransmitter and changes its volume when challenged with small ionic concentration change. Immunodetection analysis revealed that the synaptic vesicle proteoglycan SV2 is the core of the intravesicular matrix and is responsible for immobilization and release of ACh and ATP. We suggest that in the early steps of vesicle fusion, this internal matrix regulates the availability of free diffusible ACh and ATP, and thus serves to modulate the quantity of transmitter released.

Release of ATP induced by hypertonic solutions in Xenopus oocytes.

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mM). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water.

Calcium-dependent acetylcholine release from Xenopus oocytes: simultaneous ionic currents and acetylcholine release recordings.

The fusion of synaptic vesicles with presynaptic membranes is controlled by a complex network of protein-protein and protein-lipid interactions. SNAP-25, syntaxin and synaptobrevin (SNARE complex) are thought to participate in the formation of the core of the membrane fusion machine but the molecular basis of SNARE interactions is not completely understood. Thus, it would be interesting to design experiments to test those relationships in a new model. Xenopus laevis oocytes are valuable tools for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Here we show that SNARE proteins are present in native Xenopus oocytes and that those oocytes injected with acetylcholine and presynaptic plasma membranes extracted from the electric organ of Torpedo marmorata assume some of the functions of a cholinergic nerve terminal. Neurotransmitter release and macroscopic currents were recorded and analysed simultaneously in a single oocyte electrically depolarized: acetylcholine release was detected using a chemiluminiscent method and calcium entry was measured by exploiting the endogenous Ca2+-activated chloride current of the oocyte with a two-electrode voltage-clamp system. Neurotransmitter release was calcium- and voltage-dependent and partially reduced in the presence of several calcium channel blockers. Clostridial neurotoxins, both holotoxin and injected light-chain forms, also inhibited acetylcholine release. We also studied the role of the SNARE complex in synaptic transmission and membrane currents by using monoclonal antibodies against SNAP-25, syntaxin or VAMP/synaptobrevin. The use of antibodies against VAMP/synaptobrevin, SNAP-25 and syntaxin inhibited acetylcholine release, as did clostridial toxins. However, macroscopic currents were only modified either by syntaxin antibody or by Botulinium-C1 neurotoxin. This model constitutes a new approach for understanding the vesicle exocytosis processes.