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The article discusses the use of total internal reflection fluorescence microscopy (TIRFM) to evaluate the glutamatergic signaling in the nervous system and in peripheral organs. Topics include the dynamic modulation of glutamate transporter density, the molecular mechanisms that control excitatory amino acid carrier 1 (EAAC1) surface density, and the mechanisms of allopregnanolone (ALLO).

Abstract Background Emerging evidence indicates that, similarly to what happens for peripheral macrophages, microglia can express different phenotypes depending on microenvironmental signals. In spite of the large literature on inflammation after ischemia, information on M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is lacking. The present study investigates the presence of microglia/macrophage phenotype markers, their temporal expression, whether they are concomitantly expressed by the same subpopulation, or they are expressed at distinct phases or locations in relation to the ischemic lesion. Methods Volume of ischemic lesion, neuronal counts and TUNEL staining were assessed in C57Bl/6 mice at 6-12-24-48 h and 7d after permanent occlusion of the middle cerebral artery. At the same time points, the expression, distribution in the lesioned area, association with a definite morphology and coexpression of the microglia/macrophage markers CD11b, CD45, CD68, Ym1, CD206 were assessed by immunostaining and confocal microscopy. Results The results show that: 1) the ischemic lesion induces the expression of selected microglia/macrophage markers that develop over time, each with a specific pattern; 2) each marker has a given localization in the lesioned area with no apparent changes during time, with the exception of CD68 that is confined in the border zone of the lesion at early times but it greatly increases and invades the ischemic core at 7d; 3) while CD68 is expressed in both ramified and globular CD11b cells, Ym1 and CD206 are exclusively expressed by globular CD11b cells. Conclusions These data show that the ischemic lesion is accompanied by activation of specific microglia/macrophage phenotype that presents distinctive spatial and temporal features. These different states of microglia/macrophages reflect the complexity of these cells and their ability to differentiate towards a multitude of phenotypes depending on the surrounding micro-environmental signals that can change over time. The data presented in this study provide a basis for understanding this complex response and for developing strategies resulting in promotion of a protective inflammatory phenotype.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect. [ABSTRACT FROM AUTHOR]

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside-out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X-100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high-resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high-resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins. [ABSTRACT FROM AUTHOR]

Abstract: The effect of operating conditions (temperature, space velocity, pressure and H2/feed ratio) on the hydroconversion of Fischer–Tropsch wax and isomer content of the middle distillate was investigated by carrying out experiments according to a second order factorial design. The catalyst used was a 0.3% platinum/amorphous silica–alumina catalyst. Temperature and space velocity exhibited the biggest effect on wax conversion rate but a significant role was played by the pressure and the H2/wax ratio as well. The results indicated an inverse relationship between pressure and conversion rate while an opposite behaviour was shown by the H2/wax ratio. The effect of the last two variables was explained respectively in terms of the reaction mechanism of n-paraffins on bifuctional catalyst and the effect of the vapour–liquid equilibrium (VLE) on apparent conversion rate and isomer content of products. [Copyright &y& Elsevier]

This work describes the new facility for applied nuclear physics at the University of Sao Paulo, mainly for irradiation of electronic devices. It is a setup composed of a quadrupole doublet for beam focusing/defocusing plus multiple scattering through gold foils to produce low intensity, large-area, and high-uniformity heavy-ion beams from 1H to 107Ag. Beam intensities can be easily adjusted from 1 0 2 p a r t i c l e s c m 2 / s to hundreds of nA for an area as large as 2.0 c m 2 and uniformity better than 90%. Its irradiation chamber has a high-precision motorized stage, and the system is controlled by a LabViewTM environment, allowing measurement automation. Design considerations and examples of use are presented. [ABSTRACT FROM AUTHOR]

The article discusses a study showing increase in acute stress due to readily releasable glutamate vesicles in prefrontal and frontal cortex by the non-genomic action of corticosterone.

Abstract: The reactivity of Mg/Me-mixed oxides (Me=Fe, Cr, Al) in m-cresol gas-phase methylation with methanol was studied, with the aim of finding relationships between catalytic performance and surface/bulk features. All systems exhibit similar surface properties (basically due to the presence of basic and Lewis-type acid sites), but the presence of extra cations in the MgO framework affects greatly the products obtained in the mentioned catalytic reaction, depending on the possible co-presence of redox capacity. In particular, (i) the Mg/Al/O system, characterized by no redox capacity, shows catalytic behaviour quite similar to that of conventional Brønsted-type acid catalysts, though with the advantage of exhibiting almost no deactivation during use (a rather common problem for acid catalysts activating methanol for phenol attack); (ii) the Mg/Cr/O catalyst shows a reactivity typical of a basic catalyst, with high chemo- and regio-selectivity and the favoured formation of 2,5-dimethylphenol; (iii) the Mg/Fe/O system presents a catalytic behaviour closer to that of Mg/Cr/O, if referred to the distribution of phenolic products, but it also exhibits a better activity in methanol dehydrogenation to formaldehyde (that is the true electrophilic reactant for m-cresol ring methylation), and in the decomposition of formaldehyde into light compounds. This behaviour causes a poor selectivity to dimethylphenols with respect to converted methanol, whereas the selectivity with respect to converted m-cresol is not different from that shown by Mg/Cr/O system. [Copyright &y& Elsevier]

Abstract: The alkylation of phenol with methanol was studied using a Brønsted-type acid catalyst (a H-mordenite) and basic/dehydrogenating catalysts (MgO, Fe2O3 and Mg/Fe/O), with the aim of investigating the reaction mechanism. The main difference between the two classes of catalysts concerned the transformations occurring on methanol. Specifically, in the former case the acid-type activation of methanol led to the development of an electrophylic species that gave rise to the formation of anisole and of C-alkylated compounds. With basic catalysts, methanol dehydrogenated to formaldehyde, which then underwent transformation to methylformate and to decomposition products, i.e., CO, CO2, CH4 and H2. In this case, the prevailing compounds obtained by reaction with phenol were o-cresol and 2,6-xylenol. The dehydrogenation of methanol was found to be the key-step in the generation of the active methylating species with basic catalysts. [Copyright &y& Elsevier]

We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter. [ABSTRACT FROM AUTHOR]

A double superconducting solenoid system is being installed at the Pelletron Laboratory of the University of S ao Paulo, Brazil. This system allows the production of secondary beams of light exotic nuclei like 8Li, 6He and others. The first results using this facility are presented. [ABSTRACT FROM AUTHOR]

Magnesium/aluminium mixed oxides were synthesised following two procedures, via: (i) coprecipitation (CP) and (ii) sol–gel (SG). A crystalline precursor with hydrotalcite-like structure developed in both cases only for Mg/Al ratios between 2 and 4. The thermal treatment of the precursors yielded the mixed oxides, which had different bulk and surface properties depending on the preparation method and the Mg/Al ratio. The calcined materials were tested in the liquid-phase alkylation of m-cresol with methanol. Relationships were found between the basic characteristics and the catalytic performance. In the range of Mg/Al ratios between 2 and 4, SG oxides had a higher degree of crystal phase than the CP ones, and different morphology. This difference corresponded to differences also in the surface basic features, which however did not influence considerably the catalytic performances. When low Mg/Al ratios were used, the differences between SG and CP samples were greater. Specifically, the CP procedure favoured segregation of oxides and formation of a large number of strong basic sites. However these sites soon deactivated under reaction conditions and did not directly contribute to the reaction. In the case of the SG procedure, a more efficient reciprocal dispersion of the two elements was achieved, with generation of specific sites of medium basic strength, which affected the catalytic performance. SG materials with low Mg/Al ratio (Mg/Al=0.15) showed an improvement of the methylation regioselectivity (e.g., decrease of the O/C-alkylation ratio and increase of the ortho/para-C-alkylation ratio) with respect to both CP and SG samples having higher Mg/Al ratios. [Copyright &y& Elsevier]

The side-chain alkylation of toluene with methanol is one alternative technology to produce styrene that has been given attention in the last few years. In the literature basic materials has been proposed as catalyst for the reaction but the real number of tested catalysts is very small and few preparation parameters have been taken into account. In this work a combinatorial approach has been used to explore the possibilities of basic zeolites to carry out such reaction. To do this, the following catalyst variables have been studied: nature of zeolite, framework composition, nature and content of compensating cation and method of incorporation (exchange, impregnation). The results obtained confirm the requirements of both basic and acid sites in the catalysts and show the compromise between these two functions. The study carried out shows that zeolite-based catalysts are still poor reactive to give the styrene/ethylbenzene yields required for converting this process in a real alternative to the existing one, based on the alkylation of benzene with ethylene, followed by the dehydrogenation of ethylbenzene to styrene. [Copyright &y& Elsevier]

Sympathetic hyperactivity in rats with heart failure is associated with increased extracellular noradrenaline in the hypothalamic paraventricular nucleus at rest. However, it is unknown how this nucleus responds to stressful stimuli. In the present study we therefore examined the basal and stress-induced release of noradrenaline in the paraventricular nucleus of conscious Sprague–Dawley rats with heart failure measured by in vivo microdialysis. Basal noradrenaline concentration in the paraventricular nucleus of rats with heart failure was more than double that in sham-operated controls. Immobilization stress decreases noradrenaline levels in the paraventricular nucleus of rats with heart failure to 57% of baseline, while it increased in sham-operated controls to 228%. However, serum corticosterone was similarly elevated at 30 and 90 min post-stress in both experimental groups.We have shown that heart failure causes an impairment of the central noradrenergic system’s response to acute sympatho-excitation but does not affect the hypothalamo–pituitary–adrenocortical response. [Copyright &y& Elsevier]

Mg/Al mixed oxides, obtained by decomposition of hydrotalcite-like precursors, represent interesting heterogeneous catalytic systems for basic-catalyzed reactions, as an alternative to environmentally unfriendly homogeneous catalysts. The reactivity of these oxides was evaluated using the methylation of m-cresol as a test reaction and relationships between catalytic performance and chemical–physical features were established. The basicity of the samples was evaluated by CO2 adsorption and thermal-programmed-desorption. The presence of Al in the mixed oxides considerably affected the density and the strength of the basic sites with respect to MgO. These basic properties in turn influenced the catalytic performance of the materials. Under the reaction conditions used in the present work, medium strength basic sites played the major role in the reaction. [Copyright &y& Elsevier]

The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2−/−), ficolin-A (Fcna−/−), CL-11 (Colec11−/−), MASP-1/3 (Masp1−/−), MBL-C (Mbl2−/−), MBL-A (Mbl1−/−) or MBL−/− (Mbl1−/−/Mbl2−/−) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2−/−, MBL−/− and FCN-A−/− mice had better outcome scores compared to WT. Of these, MASP-2−/− mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2−/− mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI. [ABSTRACT FROM AUTHOR]