Prevention of excessive exercise-induced adverse effects in rats with Bacillus subtilis BSB3.

AIMS: To characterize efficacy of the Bacillus subtilis BSB3 (BSB3) strain in the prevention of excessive exercise-induced side effects and in maintaining stability of the gut microbiota. METHODS AND RESULTS: Rats were pretreated by oral gavage with B. subtilis BSB3 (BSB3) or with phosphate-buffered saline (PBS) twice a day for 2 days, and were either exposed forced treadmill running or remained sedentary. Histological analysis of intestine, immunofluorescence staining of tight junction (TJ) proteins, serum lipopolysaccharide and intestinal fatty acid-binding protein assay, culture-based analysis and pyrosequencing for the gut microbiota were performed for each rat. Forced running resulted in a substantial decrease in intestinal villi height and total mucosa thickness, the depletion of Paneth cells, an inhibition of TJ proteins expression. Short-term treatment of rats with BSB3 before running prevented these adverse effects. Culture-based analysis of the gut microbiota revealed significant elevation of pathogenic microorganisms only in treadmill-exercised rats pretreated with PBS. High-throughput 16S rRNA gene sequencing also revealed an increase in pathobionts in this group. Preventive treatment of animals with BSB3 resulted in predominance of beneficial bacteria. CONCLUSIONS: BSB3 prevents excessive exercise-associated complications by beneficial modulation of the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study shows a new application of beneficial bacteria for prevention the adverse effects of excessive exercise.

Yeast fermentate prebiotic improves intestinal barrier integrity during heat stress by modulation of the gut microbiota in rats.

AIMS: To evaluate efficacy of Saccharomyces cerevisiae fermentate prebiotic (EH) in protection of intestinal barrier integrity in rats during heat stress, to analyze the impact of heat stress and preventive treatment with EH on the structure of the gut microbiota. METHODS AND RESULTS: Two groups of rats were treated orally with EH or phosphate-buffered saline for 14 days. On day 15, half of the rats in each group were exposed to heat stress conditions, while control animals were kept at room temperature. Histological and Western blot analyses of the intestine, culture-based microbiological analysis and high-throughput 16S rRNA sequencing for the gut microbiota were performed for each rat. Exposure of animals to heat stress conditions resulted in inhibition of tight junction (TJ) proteins expression, decrease of Paneth and goblet cells, decrease of beneficial and increase of pathogenic bacteria. Oral treatment of rats with EH before stress significantly prevents these adverse effects by elevation of the gut beneficial bacteria, particularly butyrate-producing bacteria. CONCLUSIONS: Essential effect of EH in protection of intestinal barrier integrity during heat stress is connected with beneficial modulation of the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results will contribute to the development of new approaches to prevention of heat stress-related complications.

Thermodynamic evaluation of vesicles shed by erythrocytes at elevated temperatures.

Erythrocytes undergo structural transformation and shed small vesicles at elevated temperatures. To characterize mechanisms of the phenomenon, the Arrhenius and Eyring equations were used for analysis of the literature, experimental data on vesiculation of human and rat erythrocytes after the temperature was elevated by physical exercise or by exposure to external heat. The thermodynamic analysis of the data showed that erythrocyte transformation, vesicle release, and other associated processes are driven by entropy with enthalpy-entropy compensation. It is suggested that the physical state of the hydrated cell membrane is responsible for the compensation. The increase of vesicle number related to elevated temperatures may be indicative of the heat stress level and serve as diagnostic of erythrocyte stability and human performance.

Oral administration of Bacillus subtilis strain BSB3 can prevent heat stress-related adverse effects in rats.

AIMS: To determine the efficacy of Bacillus subtilis strain in prevention of heat stress-related complications in rats. METHODS AND RESULTS: Male Sprague-Dawley rats weighing 250-300 g were treated by oral gavage with B. subtilis BSB3 strain or PBS twice a day for 2 days. Half of the rats of each group were exposed to heat stress (45°C, relative humidity 55% for 25 min), while the remaining rats were placed at identical conditions but at 25°C. Bacterial translocation, histological changes in the intestine, cytokines profile, serum lipopolysaccharide level (LPS), as well as vesiculation of erythrocytes were analysed and compared between groups. Adverse effects of heat stress (morphological changes in intestine, bacterial translocation, elevated levels of LPS and IL-10, increased vesiculation of erythrocytes) were observed only in rats not protected with B. subtilis strain and exposed to heat. All registered parameters in rats pretreated with bacilli and exposed to heat were similar to control groups. CONCLUSIONS: Bacillus subtilis BS3B strain was effective in the prevention of complications related to heat stress in rats. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute towards better understanding of probiotics' mechanisms of action and will bring new approaches to characterize and use of beneficial bacteria.

Rapid and sensitive magnetoelastic biosensors for the detection of Salmonella typhimurium in a mixed microbial population.

In this article, we report the results of an investigation into the performance of a wireless, magnetoelastic biosensor designed to selectively detect Salmonella typhimurium in a mixed microbial population. The Langmuir-Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors (2 x 0.4 x 0.015 mm). Bacterial binding to the antibody on the sensor surface changes the resonance parameters, and these changes were quantified as a shift in the sensor's resonance frequency. Response of the sensors to increasing concentrations (5 x 10(1) to 5 x 10(8) cfu/ml) of S. typhimurium in a mixture of extraneous foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was studied. A detection limit of 5 x 10(3) cfu/ml and a sensitivity of 139 Hz/decade were observed for the 2 x 0.4 x 0.015 mm sensors. Binding kinetics studies have shown that the dissociation constant (K(d)) and the binding valencies for water samples spiked with S. typhimurium was 435 cfu/ml and 2.33 respectively. The presence of extraneous microorganisms in the mixture did not produce an appreciable change in the biosensor's dose response behavior.

Structure and function of long-lived olfactory organotypic cultures from postnatal mice.

The first synapse in the olfactory pathway mediates a significant transfer of information given the restricted association of specific olfactory receptor neurons with specific glomeruli in the olfactory bulb. To understand better how this connection is made and what the functional capacities of the participating cells are, we created a long-lived culture system composed of olfactory epithelium and olfactory bulb tissues. Using the roller tube method of culturing, we grew epithelium-bulb cocultures, explanted from 1-4-day-old Swiss Webster mice, on Aclar for periods ranging from 18 hr to 68 days. The explants flattened so that in some areas the culture was only a few cells thick, making individual cells distinguishable. From 107 cultures studied, we identified the following cell types by expression of specific markers (oldest culture expressing marker, days in vitro, DIV): olfactory receptor neurons (neural cell adhesion molecule, 42 DIV); mature receptor neurons (olfactory marker protein, 28 DIV); postmitotic olfactory receptor neurons and olfactory bulb neurons (beta-tubulin, 68 DIV); astrocytes (glial fibrillary acidic protein, glutamate/aspartate transporter, 68 DIV); olfactory horizontal basal cells (cytokeratin, 22 DIV). Neuronal processes formed glomeruli in 2-4-week-old cultures. We also recorded electro-olfactography responses to puffs of vapor collected over an odorant mixture containing ethyl butyrate, eugenol, (+) carvone, and (-) carvone from cultures as old as 21 DIV. These features of our olfactory culture system make this model useful for studying properties of immature and mature olfactory receptor neurons, pathfinding strategies of receptor axons, and mechanisms of information transfer in the olfactory glomerulus.

Specific and selective biosensor for Salmonella and its detection in the environment.

The specific and selective detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated in liquid samples. These biosensors were selective to S. typhymurium in the presence of large concentrations of Escherichia coli O157:H7. They were also specific to S. typhymurium since bacteria preincubated with free antibody produced no signal. Dark-field and electron microscopy showed that two different antibodies, polyvalent somatic O and flagellar H7, were immobilized on the sensor surface producing two distinct attachments of bacteria at the liquid-solid interface. The somatic O antibody exhibits a rigid, binding, while the flagellar H7 antibody forms a flexible connection allowing a large degree of freedom. When the attachment of bacteria was rigid and strong, the responses of the acoustic wave sensors correlated with changes in the mass of bacteria present at the liquid-solid interface. In contrast, when attachment was flexible, the sensor signals were inversely proportional to the additional mass of bound bacteria. This difference is probably determined by the interfacial viscoelasticity and by acoustic and electromagnetic coupling. The signals of environmentally aged sensors with either predominantly rigid or flexible positioning of bacteria were correlated with changes in mass at the liquid-solid interface. Sensors with O or H type of binding could be used for analytical purposes.

Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors.

Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.

RGS2 regulates signal transduction in olfactory neurons by attenuating activation of adenylyl cyclase III.

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

Rapid and sensitive biosensor for Salmonella.

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.

Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain.

Glutamate receptors specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) have been reported to interact with the highly sulfated glycosaminoglycan, heparin, and to subsequently express lower binding affinity for [3H]AMPA. The present study examined whether heparin also modifies the kinetic properties of single channel activity expressed by isolated AMPA receptors from rat forebrain. Upon application of 280 nM AMPA, the partially purified receptors reconstituted in lipid bilayers expressed bursting channel activity that was inhibited by dinitroquinoxaline-2-3,-dione (DNQX). Treating the receptors with heparin (10 microg/ml) produced no change in conductance but the mean burst length for 280 nM AMPA was nearly doubled. Heparin also prolonged the lifetime of open states of the individual ion channels 3-5-fold, perhaps by causing a decrease in the closing rate constant for channel gating. Heparin had no effect on the lifetime of the closed state or on the amplitude of currents. The single channel open time was voltage-dependent and an increase of applied voltage caused a decrease in the heparin effect on channel open times. While the lifetime of the open channel was increased 3-4 times by heparin at 20 mV, there was no significant change induced at 43 mV. The equivalent electric charge of the channel gate was increased by 40%. The heparin effects were specific as another polysaccharide, dextran, and a monomeric constituent of heparin, glucosamine 2,3-disulfate, failed to have any effect on the receptors. These findings suggest that heparin-containing extracellular matrix components can interact with AMPA receptors and influence their functional properties.

Inhibition and enhancement of odorant-induced cAMP accumulation in rat olfactory cilia by antibodies directed against G alpha S/olf- and G alpha i-protein subunits.

The odorant-induced accumulation of cAMP can be inhibited by antibodies directed against G alpha s/olf. In contrast, antibodies raised against G alpha i-subunits caused a strong enhancement of the odorant-induced cAMP accumulation. Western blotting and immunoelectron microscopy revealed the presence of both G alpha s/olf- and G alpha i-subunits in rat cilia preparations. The existence of both stimulatory and inhibitory odorant-induced regulation of adenylyl cyclase activity in olfactory cilia may indicate that an initial integration of different odorant stimuli begins at the level of primary reactions in the same effector enzyme.

Effects of heparin on the properties of solubilized and reconstituted rat brain AMPA receptors.

Heparin was found to bind to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [3H]AMPA binding to detergent-solubilized receptors by 75% (IC50 = 10 micrograms/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [3H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [3H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 micrograms/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.

Single channel recordings of reconstituted AMPA receptors reveal low and high conductance states.

Glutamate receptors belonging to the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subclass were partially purified 30- to 60-fold from forebrain of adult rats and incorporated into planar bimolecular lipid membranes. The channel conductance associated with the reconstituted receptors was activated by kainate and AMPA in a manner that suggests cooperative binding of two to three agonist molecules is required to induce channel opening. This conductance was blocked by the specific antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). When the partially purified AMPA receptors were reconstituted by the tip-dipping method in asymmetric saline conditions ('outside-out configuration'), the addition of 300 nM AMPA to the pseudo-extracellular solution elicited single channel current fluctuations that were also inhibited by DNQX. Analyses of the currents revealed that the ion channels of reconstituted AMPA receptors have two distinct conductance levels of 12 and 60 pS with the great majority of receptors belonging to the former variety. These results suggest that reconstitution may be useful in identifying factors that regulate the binding and conductance properties of AMPA receptors.

Functional reconstitution of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors from rat brain.

Glutamate receptors belonging to the subclass specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were solubilized from rat forebrain membranes with Triton X-100 and partially purified through a series of three chromatographic steps. Specific [3H]AMPA binding increased 30-60-fold during the isolation procedure. A protein band recognized by antibodies against specific amino acid sequences of the glutamate receptor-A subunit was enriched with each purification step; the molecular mass of this band (105 kDa) corresponded to that of cloned AMPA receptor subunits. Photoaffinity labeling of forebrain membranes with 6-cyano-7-[3H]nitroquinoxaline-2,3-dione, a specific antagonist of the AMPA receptor, labeled a single band that comigrated with the immunolabeled protein. On reconstitution of the partially purified material into bilayer patches, single-channel current fluctuations were elicited by 300 nM AMPA and blocked by 1 microM 6,7-dinitroquinoxaline-2,3-dione.

Surface properties of two rabbit lung lamellar body preparations with markedly different fatty acid profiles.

The effect of fatty acid desaturation on the surface properties of lung surfactant were studied on a Wilhelmy surface balance by using two preparations of lamellar body (LB) material with markedly different fatty acid profiles: (1) lamellar bodies from adult rabbit lung tissue, and (2) lamellar bodies from fetal rabbit lung tissue maintained in organ culture for 7 days. The fetal lung preparation contains an unusually high level of 16: 1 fatty acid (principally palmitoleic acid) at position sn-2 of phosphatidylcholine (Longmuir, K.J., Resele-Tiden, C. and Rossi M.E. (1988) J. Lipid Res. 29, 1065-1077). Surface pressure-surface area isotherms were obtained for both preparations and compared to isotherms of monolayers of dipalmitoylphosphatidylcholine. In addition, the elasticity of the lamellar body preparations were analyzed as a function of surface pressure, temperature, and rate of compression, both in the presence and absence of Ca2+ plus Mg2+. At slow rates of compression, we found that fetal LB films have lower elasticity and better respreading ability compared to the adult LB films, which can be explained by the high concentration of unsaturated palmitoleic acid in the fetal preparation. A dynamic component of elasticity was observed at high rates of compression only if Ca2+ and Mg2+ were present in the subphase. The analysis of the free energies, enthalpies and entropies of compression suggests that films with low concentrations of unsaturated fatty acids are are likely to undergo irreversible collapse, but films with excess unsaturated fatty acids accommodate the overcompression with a reversible loss of molecules from the surface.

Alamethicin adsorption to a planar lipid bilayer.

The effect of alamethicin and its derivatives on the voltage-dependent capacitance of phosphatidylethanolamine (squalane) membranes was measured using two different methods: lock-in detection and voltage pulse. Alamethicin and its derivatives modulate the voltage-dependent capacitance at voltages lower than the voltage at which alamethicin-induced conductance is detected. The magnitude and sign of this alamethicin-induced capacitance change depends on the aqueous alamethicin concentration and the kind of alamethicin used. Our experimental data can be interpreted as a potential-dependent pseudocapacitance associated with adsorbed alamethicin. Pseudocapacitance is expressed as a function of alamethicin charge, its concentration in the bathing solution and the applied electric field. The theory describes the dependence of the capacitance on applied voltage and alamethicin concentration. When alamethicin is neutral the theory predicts no change of the voltage-dependent capacitance with either sign of applied voltage. Experimental data are consistent with the model in which alamethicin molecules interact with each other while being adsorbed to the membrane surface. The energy of this interaction depends on the alamethicin concentration.

Functional reconstitution of N-methyl-D-aspartate receptors in artificial lipid bilayers.

Functional reconstitution of the N-methyl-D-aspartate (NMDA) receptors was achieved by adding synaptic membranes from rat brain to large planar bimolecular lipid membranes (BLMs). The reconstituted receptors exhibited several properties of the NDMA receptors described using a variety of biochemical and electrophysiological techniques. Addition of NMDA at concentrations between 5 and 50 microM produced large, voltage-dependent increases in membrane conductivity. The selective antagonist of NMDA receptors, amino-2-phosphonopentanoate (AP-7), totally blocked the response of the bilayers to NMDA as did micromolar concentrations of magnesium; this latter effect was also voltage-dependent. These results indicate that BLMs can be used to study the ion channels and regulatory processes associated with NMDA receptors from adult brain in ways that could not be accomplished with conventional neurophysiological techniques.

ATP and GTP are essential for olfactory response.

The steady-state conductance of planar bimolecular lipid membranes (BLMs) modified with rat olfactory epithelial homogenate (ROH) becomes sensitive to very low concentrations of odorant in the presence of adenosine triphosphate (ATP) and guanosine triphosphate (GTP). The chemosensitivity is not observed when ATP and GTP are absent. Adenosine 3',5'monophosphate (cAMP) mimics the effect of the odorant. Effects of odorants and cAMP are dose dependent. These data are consistent with the hypothesis that cAMP is a second messenger in the initial steps of olfactory transduction.

Current-voltage characteristics of planar lipid membranes with attached Halobacterium cell-envelope vesicles.

We have studied the photoactivity of a system consisting of large, planar, essentially solvent free bilayers bearing adsorbed cell-envelope vesicles prepared from Halobacterium halobium (strain L 33). The system was made conductive by addition of a proton carrier (SF-6847). We observed photocurrents which were linearly dependent upon transmembrane voltage. Current-voltage curves were found to be well described by an equivalent circuit with the following significant parameters: planar bilayer conductance, planar bilayer-vesicle contact area conductance, cell-envelope vesicle conductance, and chloride pump equivalent voltage-generator potential. These parameters are uniquely obtained as a result of a few independent current measurements. The stationary photovoltage was dependent upon chloride concentration, and from this dependence an active transport (pump) affinity of the system for chloride was calculated to be about 50 mM.