Symbiosis with systemic fungal endophytes promotes host escape from vector-borne disease.

Plants interact with a myriad of microorganisms that modulate their interactions within the community. A well-described example is the symbiosis between grasses and Epichloë fungal endophytes that protects host plants from herbivores. It is suggested that these symbionts could play a protective role for plants against pathogens through the regulation of their growth and development and/or the induction of host defences. However, other endophyte-mediated ecological mechanisms involved in disease avoidance have been scarcely explored. Here we studied the endophyte impact on plant disease caused by the biotrophic fungus, Claviceps purpurea, under field conditions through (1) changes in the survival of the pathogen´s resistance structure (sclerotia) during overwintering on the soil surface, and (2) effects on insects responsible for the transportation of pathogen spores. This latter mechanism is tested through a visitor exclusion treatment and the measurement of plant volatile cues. We found no significant effects of the endophyte on the survival of sclerotia and thus on disease inocula. However, both pathogen incidence and severity were twofold lower in endophyte-symbiotic plants than in non-symbiotic ones, though when insect visits were prevented this difference disappeared. Endophyte-symbiotic and non-symbiotic plots presented different emission patterns of volatiles suggesting that they can play a role in this protection. We show a novel indirect ecological mechanism by which endophytes can defend host grasses against diseases through negatively interacting with intermediary vectors of the epidemic process.

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

Fourier transform infrared difference spectra of intermediates in rhodopsin bleaching.

The membrane protein rhodopsin is the primary light receptor in vision. Fourier transform infrared difference spectroscopy is sensitive to conformational changes in both the protein and the retinylidene chromophore of rhodopsin. By blocking rhodopsin bleaching at specific intermediates, it is possible to elucidate some of the primary molecular events of vision.

No Vertical Stratification Found in Cavity-Nesting bees and Wasps in Two Neotropical Forests of Argentina.

Vertical stratification is known in diverse arthropod communities in forests, but little is known about nesting ecology of trap-nesting bees and wasps in Neotropical forests. We studied the vertical colonization of trap-nests by solitary bees and wasps in two forests in Argentina. We obtained 204 nests of seven bee and six wasp species, with 1040 brood cells from which 660 hosts and 32 parasites (21 parasitoid and 11 kleptoparasite) adults emerged from four groups (five species of wasps [Chrysididae, Eulophidae, and Ichneumonidae], four flies [Bombyliidae and Sarcophagidae], three bees [Apidae and Megachilidae], and one species of beetle [Meloidae]). The number of nests, reproductive success, parasitism rate, and mortality did not differ between canopy and understory, nor did the number of brood cells, emerged adults per trap-nest, and total abundance per transect. We found similar assemblages of trap-nesting bees and wasps in both forests, but contrary to our expectations, we did not find any significant difference in the analyzed variables. Our results suggest that access to resources used by trap-nesting bee and wasp females was at least similar, and/or that the microclimatic conditions were homogeneous in canopy and understory. Moreover, our results agree with those reported by some researchers but contrast with others, demonstrating the great variation in the response of bees and wasps nesting in preexisting cavities to vertical stratification in forests and the need for more studies about this topic.

Opioid receptor modulation of a metabolically sensitive ion channel in rat amygdala neurons.

We have used single-channel patch-clamp recordings to study opiate receptor effects on freshly dissociated neurons from the rat amygdalohippocampal area (also called the posterior nucleus of the amygdala), an output nucleus of the amygdala implicated in appetitive behaviors. Dissociated cells included a distinct subpopulation that was 30-40 micrometer in diameter, multipolar or pyramidal in shape, and immunoreactive for neuron-specific enolase, mu opioid receptors, and galanin. In whole-cell perforated-patch recordings, these cells responded to low concentrations of mu opioid agonists with a hyperpolarization. In cell-attached single channel recordings, these cells expressed a large variety of K(+)-permeable ion channels, including 20-100 pS inward rectifiers and 150-200 pS apparent Ca(2+)-activated K(+) channels, none of which appeared sensitive to the presence of opioid drugs. In contrast, a 130 pS inwardly rectifying channel was selectively activated by mu opioid receptors in this same subpopulation of cells and was active only in the presence of opioid agonists, and inhibited in the presence of antagonists. Channels identical to the 130 pS channel in conductance and voltage sensitivity were activated in the absence of opioids, when the cells were treated with glucose-free medium or with the metabolic inhibitor rotenone. The sulfonylurea drug tolbutamide inhibited 130 pS channel openings elicited by opioids. Thus, a subpopulation of amygdala projection neurons expresses a metabolically sensitive ion channel that is selectively modulated by opiate receptors. This mechanism may allow opioid neurotransmitters to regulate ingestive behaviors, and thus, opiate drugs to influence reward pathways.

Facilitation of sodium currents in frog neuroglia by nerve impulses: dependence on external calcium.

Facilitation of voltage-gated sodium currents in glial membranes by nerve impulses has been studied by using both the whole cell and loose-patch clamp techniques in the isolated intact optic nerve of the frog. During facilitation there is a shift in the voltage dependence of glial Na+ channels such that a given depolarization produces a larger inward Na+ current. Decreasing external calcium from 4 times normal to 0.2 times normal produced a similar shift in the current-voltage relation. Increasing the external calcium concentration to 4-5 times normal blocks facilitation. In reduced calcium, 0.1-0.2 times normal, the peak of facilitation was unaffected, but its decay was slowed. The addition of 1 mM nickel and 2 mM cobalt or 2 mM cadmium, to prevent depletion of extracellular calcium that might result from voltage-dependent entry of calcium into the axons, did not block the facilitation. The results suggest that, even though facilitation is blocked by high extracellular calcium, a decrease in extracellular calcium produced by axon impulses is not the cause of the facilitation.

Cardiovascular autonomic function in anthracycline-treated breast cancer patients.

Preclinical studies suggest that in addition to the well-known direct damage to the myocardium, anthracycline antineoplastic drugs exert toxic effects on the cardiovascular autonomic system as well. To investigate whether this phenomenon occurs in the clinic, we carried out noninvasive, widely used tests of cardiovascular autonomic physiology in 55 women with stage II or III breast cancer. In all, 31 were being treated with anthracycline-containing chemotherapy regimens, and 24 who were receiving CMF (cyclophosphamide, Methotrexate, and fluorouracil) served as controls. Of 279 tests conducted in anthracycline (A)-treated patients, 123 were abnormal, vs 54 of 216 tests carried out in 24 controls (44% vs 25%; P less than 0.005). Abnormal variations in heart rate on standing and in diastolic blood pressure during handgrip was found in 25 (81%) and 17 patients receiving A, vs 9 (37%; P less than 0.005) and 5 (21%; P less than 0.0001), respectively, in controls. The incidence of abnormal tests was significantly higher in A-treated patients greater than 60 years of age (41%) vs 67%; P less than 0.05). Radionuclide ventriculography was carried out in 19 patients who showed abnormal tests of cardiovascular autonomic function after greater than or equal to 6 courses of a-containing chemotherapy; only 1 of them had abnormal cardiac contractility (global hypokinesia), suggesting that abnormal tests of cardiovascular autonomic function may occur in the absence of a detectable deterioration in left ventricular ejection fraction. A large number of factors may alter cardiovascular autonomic function in cancer patients, including age, radiation therapy to the chest, and multidrug treatment. Even after correcting for the most obvious of these, chemotherapy with anthracyclines is associated with a significantly higher percentage of abnormal tests for cardiovascular autonomic function. Although indirect and semi-quantitative, our results are compatible with the idea of A-induced cardiac autonomic dysfunction.

Voltage-gated currents recorded from the surface of the frog optic nerve.

Macroscopic voltage-dependent currents were recorded from the surface of the intact optic nerve of Rana pipiens using the loose patch clamp technique. Depolarizing steps of more than 40 mV produced sodium-dependent TTX sensitive inward currents and a 4-AP and sodium sensitive fast outward current in addition to a slower outward current. Since the surface of the nerve is a glia limitans, it appears that the membranes of these astrocytes contain both voltage-sensitive sodium and potassium channels.

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments. An increase in the intensity of peaks in the amide I and II regions agrees with recent time-resolved kinetic FTIR-difference measurements and indicates that a localized protein conformational change involving the peptide backbone of bR occurs which is not evident at the lower temperature.

Nerve impulses increase glial intercellular permeability.

Coordinating the activity of neurons and their satellite glial cells requires mechanisms by which glial cells detect neuronal activity and change their properties as a result. This study monitors the intercellular diffusion of the fluorescent dye Lucifer Yellow (LY), following its injection into glial cells of the frog optic nerve, and demonstrates that nerve impulses increase the permeability of interglial gap junctions. Consequently, the spatial buffer capacity of the neuroglial cell syncytium for potassium, other ions, and small molecules will be enhanced; this may facilitate glial function in maintaining homeostasis of the neuronal microenvironment.

Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates.

It is possible, by using Fourier-transform infrared (FTIR) difference spectroscopy, to detect the conformational changes occurring in both the protein and the chromophore of bacteriorhodopsin during the photocycle. In contrast to Raman spectroscopy, a laser is unnecessary and hence the problem of a perturbing probe beam is eliminated. Furthermore, the relatively high signal-to-noise ratio obtainable with FTIR enables measurements to be made in minutes over a large spectral range. In the study reported in this paper, we used this method to examine the state of protonation of the retinylidene Schiff base in light-adapted bR570 and in K, the first intermediate in the photocycle. Resonance Raman spectroscopy provides evidence that bR570 is protonated, but these results have been questioned on the basis of theoretical and experimental grounds. FTIR difference spectral changes in the bR570-to-K transition clearly indicate that bR570 contains a protonated Schiff base. In contrast, the K intermediate displays a Schiff base that is altered but still is associated to some degree with a proton. Because the low-temperature FTIR difference spectrum of bR570 and K is similar to the recently reported low-temperature resonance Raman spectra of bR570 and K [Braiman, M. & Mathies, R. (1982) Proc. Natl. Acad. Sci. USA 79, 403-407], we can assign most, but not all, vibrational changes in the bR570-to-K transition to the chromophore. These results are consistent with a simple model of the first step in the photocycle which involves a movement of the Schiff base proton away from a counterion.

Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta.

The antennal lobe of the moth contains several classes of glial cells that are likely to play functional roles in both the developing and mature lobe. In this study, confocal and electron microscopy were used to examine in detail the morphology of two classes of glial cells, those associated with olfactory receptor axons as they course to their targets in the lobe and those that form borders around the synaptic neuropil of the olfactory glomeruli. The former, the nerve-layer glia, have long processes with multiple expansions that enwrap axon fascicles; the latter, the neuropil glia, constitute two subgroups: complex glia with large cell bodies and branching, vellate arbors; and simple glia, with multiple, mostly unbranched processes with many lamellate expansions along their lengths. The processes of complex glia appear to be closely associated with axon fascicles as they enter the glomeruli, while those of the simple glia surround the glomeruli as part of a multi-lamellar glial envelope, their processes rarely invading the synaptic neuropil of the body of the glomerulus. The full morphological development of antennal-lobe glial cells requires more than two-thirds of metamorphic development. During this period, cells that began as cuboidal or spindle-shaped cells that were extensively dye-coupled to one another gradually assume their adult form and, at least under nonstimulated conditions, greatly reduce their coupling. These changes are only weakly dependent on the presence of olfactory receptor axons. Glial processes are somewhat shorter and less branched in the absence of these axons, but basic structure and degree of dye-coupling are unchanged.

Altered gating of opiate receptor-modulated K+ channels on amygdala neurons of morphine-dependent rats.

The molecular mechanism of tolerance to opiate drugs is poorly understood. We have used single-channel patch-clamp recordings to study opiate receptor effects on dissociated neurons from rat amygdala, a limbic region implicated in addiction processes. A 130-pS inwardly rectifying K(+)-preferring cation channel was activated by mu opioid receptors in a membrane-delimited manner. After chronic treatment of the rats with morphine, channel gating changed markedly, with an approximately 100-fold decrease in open probability at a given morphine concentration. The change in channel gating correlated both in time course and in dose of morphine treatment with the development of functional opiate dependence and appeared to arise at a step after G-protein activation and before channel permeation by K(+). This decreased receptor-channel coupling appears to be large enough to account quantitatively for opiate tolerance and may represent one of the mechanisms through which tolerance occurs.

Changes in ultrastructure and voltage-dependent currents at the glia limitans of the frog optic nerve following retinal ablation.

The surface of the frog optic nerve consists of astrocytic processes separated by narrow extracellular clefts underlying a pial sheath of loose connective tissue. Macroscopic voltage dependent currents can be recorded from this surface using the loose patch-clamp technique. In this study the changes in ultrastructure and voltage dependent Na currents have been studied for up to 1 year following removal of the retina. During the first 1-4 weeks, many of the myelinated and unmyelinated axons of the retinal ganglion cells degenerate, and the debris is phagocytosed by macrophages and glial cells. However, some morphologically intact axons remain even 12 weeks after surgery. Finally, after 16 weeks all the axons have disappeared, leaving a nerve consisting only of glial cells, some of which contain phagosomes. At 40-52 weeks after enucleation, the nerve persists, at 20-40% of the normal diameter, consisting mostly of normal looking astrocytes. The amplitude of the voltage dependent Na currents recorded from nerves during the first 1-4 weeks after enucleation, with the pial sheath intact, decreases by about 50%. After 8 weeks, the Na current recorded from the surface is about 30% of control. At 16-52 weeks after removal of the retina, when there are no intact axons, the Na current is reduced by 90%. If, however, the pial sheath is stripped away, the Na currents recorded from the glial surface are 40-50% of control during this same 16- to 52-week period, suggesting that in the all-glia nerve, the currents are shunted by the relatively thicker pial sheath.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitation of voltage-gated ion channels in frog neuroglia by nerve impulses.

The functions of glial cells in the nervous system are not well defined, with the exception of myelin production by oligodendrocytes, uptake of amino-acid synaptic transmitters, and a contribution to extracellular potassium homeostasis. Neuroglia have receptors for neurotransmitters which may be involved in neuron-glia interactions. Recent studies have demonstrated voltage-gated ion channels in glial membranes. In a study of the optic nerve of the frog, small areas of the surface were examined with the loose patch-clamp method, and voltage-gated Na+ and K+ channels, presumably located in the membranes of the astrocytes forming the glia limitans, were identified. We now report that nerve impulses in the axons of the frog optic nerve transiently alter the properties of the voltage-dependent membrane channels of the surface glial cells (astrocytes), a demonstration of a new form of neuron-glia interaction.