Leptin Targets in the Mouse Brain

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin`s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRbI-RES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor. J. Comp. Neurol. 514:518-532, 2009. (C) 2009 Wiley-Liss, Inc.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Molecular characterization of a retrotransposon in the Rhynchosciara americana genome and its association with telomere

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Brainstem oxytocinergic modulation of heart rate control in rats: effects of hypertension and exercise training

Oxytocinergic brainstem projections participate in the autonomic control of the circulation. We investigated the effects of hypertension and training on cardiovascular parameters after oxytocin (OT) receptor blockade within the nucleus tractus solitarii (NTS) and NTS OT and OT receptor expression. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were trained (55% of maximal exercise capacity) or kept sedentary for 3 months and chronically instrumented (NTS and arterial cannulae). Mean arterial blood pressure (MAP) and heart rate (HR) were measured at rest and during an acute bout of exercise after NTS pretreatment with vehicle or OT antagonist (20 pmol of OT antagonist (200 nl of vehicle)-1). Oxytocin and OT receptor were quantified (35S-oligonucleotide probes, in situ hybridization) in other groups of rats. The SHR exhibited high MAP and HR (P < 0.05). Exercise training improved treadmill performance and reduced basal HR (on average -11%) in both groups, but did not change basal MAP. Blockade of NTS OT receptor increased exercise tachycardia only in trained groups, with a larger effect on trained WKY rats (+31 +/- 9 versus +12 +/- 3 beats min-1 in the trained SHR). Hypertension specifically reduced NTS OT receptor mRNA density (-46% versus sedentary WKY rats, P < 0.05); training did not change OT receptor density, but significantly increased OT mRNA expression (+2.5-fold in trained WKY rats and +15% in trained SHR). Concurrent hypertension- and training-induced plastic (peptide/receptor changes) and functional adjustments (HR changes) of oxytocinergic control support both the elevated basal HR in the SHR group and the slowing of the heart rate (rest and exercise) observed in trained WKY rats and SHR.

In situ X-ray diffraction studies of phase transition in Pb1-x La x Zr0.40Ti0.60O3 ferroelectric ceramics

The temperature dependence of the crystalline structure and the lattice parameters of Pb1-xLaxZr0.40Ti0.60O3 ferroelectric ceramic system with 0.00 x 0.21 was determined. The samples with x 0.11 show a cubic-to-tetragonal phase transition at the maximum dielectric permittivity, Tmax. Above this amount and especially for the x = 0.12 sample, a spontaneous phase transition from a relaxor ferroelectric state (cubic phase) to a ferroelectric state (tetragonal phase) is observed upon cooling below the Tmax. Unlike what has been reported in other studies, the x = 0.13, 0.14, and 0.15 samples, which present a more pronounced relaxor behavior, also presents a spontaneous normal-to-relaxor transition, indicated by a cubic to tetragonal symmetry below the Tmax. The origin of this anomaly has been associated with an increase in the degree of tetragonality, confirmed by the measurements of the X-ray diffraction patterns. The differential thermal analysis (DSC) measurements also confirm the existence of these phase transitions.

In situ impedance spectroscopy study of the electrochemical corrosion of Ti and Ti-6Al-4V in simulated body fluid at 25 degrees C and 37 degrees C

The corrosion resistance of Ti and Ti-6Al-4V was investigated through electrochemical impedance spectroscopy, EIS, potentiodynamic polarisation curves and UV-Vis spectrophotometry. The tests were done in Hank solution at 25 degrees C and 37 degrees C. The EIS measurements were done at the open circuit potential at specific immersion times. An increase of the resistance as a function of the immersion time was observed, for Ti (at 25 degrees C and 37 degrees C), and for Ti-6Al-4V (at 25 degrees C), which was interpreted as the formation and growth of a passive film on the metallic surfaces. (C) 2009 Elsevier Ltd. All rights reserved.

Adsorption Behavior of 5-Fluorouracil on Au(111): An In Situ STM Study

The biological effects of chemical substitution of DNA bases triggered several investigations of their physicochemical properties This paper studies the adsorption behavior of a halogenated uracil, 5-fluorouracil (5FU). at the electrochemical interface of Au(111) and sulfuric acid solution. Upon modulation of the electric field across the interface, four distinct phases could be inferred by means of cyclic voltammetry (CV) At negative potentials relative to the SCE electrode, limited by the threshold of hydrogen evolution, no molecular species could be detected by scanning tunneling microscopy (STM) at the reconstructed Au(111)-(23 x root 3) surface, indicating that any physisorbed molecules are randomly distributed Incursion into more positive potentials increases the surface population but doer not form any two-dimensional (2D) physisorbed ordered structure Instead, we observed metastable structures that are only detectable. on surfaces with high defect density At sufficiently high positive potentials. limited by gold oxidation, the molecules are chemisorbed in a (3 x 2 root 3) ordered structure. with the aromatic ring perpendicular to the surface We report the densest chemisorbed monolayer for pyrimidine-derivative molecules (area per molecule 0 14 +/- 0 04 nm(2)). A comparison of the adsorption behavior of uracil derivatives has been made based on recent results of chemical substitution and solvent effects. We propose that pi-stacking is enhanced when halogens are incorporated in the uracil structure, in a similar fashion to what is observed in then crystal structure

Microgranitic Enclaves as Products of Self-mixing Events: a Study of Open-system Processes in the Maua Granite, Sao Paulo, Brazil, Based on in situ Isotopic and Trace Elements in Plagioclase

Felsic microgranular enclaves with structures indicating that they interacted in a plastic state with their chemically similar host granite are abundant in the Maua Pluton, SE Brazil. Larger plagioclase xenocrysts are in textural disequilibrium with the enclave groundmass and show complex zoning patterns with partially resorbed An-rich cores (locally with patchy textures) surrounded by more sodic rims. In situ laser ablation-(multi-collector) inductively coupled plasma mass spectrometry trace element and Sr isotopic analyses performed on the plagioclase xenocrysts indicate open-system crystallization; however, no evidence of derivation from more primitive basic melts is observed. The An-rich cores have more radiogenic initial Sr isotopic ratios that decrease towards the outermost part of the rims, which are in isotopic equilibrium with the matrix plagioclase. These profiles may have been produced by either (1) diffusional re-equilibration after rim crystallization from the enclave-forming magma, as indicated by relatively short calculated residence times, or (2) episodic contamination with a decrease of the contaminant ratio proportional to the extent to which the country rocks were isolated by the crystallization front. Profiles of trace elements with high diffusion coefficients would require unrealistically long residence times, and can be modeled in terms of fractional crystallization. A combination of trace element and Sr isotope data suggests that the felsic microgranular enclaves from the Maua Pluton are the products of interaction between end-member magmas that had similar compositions, thus recording `self-mixing` events.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (<= 36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

Feasibility of using in situ fusion for the determination of Co, Cr and Mn in Portland cement by direct solid sampling graphite furnace atomic absorption spectrometry

In situ fusion on the boat-type graphite platform has been used as a sample pretreatment for the direct determination of Co, Cr and Mn in Portland cement by solid sampling graphite furnace atomic absorption spectrometry (SS-GF AAS). The 3-field Zeeman technique was adopted for background correction to decrease the sensitivity during measurements. This strategy allowed working with up to 200 mu g of sample. The in situ fusion was accomplished using 10 mu L of a flux mixture 4.0% m/v Na(2)CO(3) + 4.0% m/v ZnO + 0.1% m/v Triton (R) X-100 added over the cement sample and heated at 800 degrees C for 20 s. The resulting mould was completely dissolved with 10 mu L of 0.1% m/v HNO(3). Limits of detection were 0.11 mu g g(-1) for Co, 1.1 mu g g(-1) for Cr and 1.9 mu g g(-1) for Mn. The accuracy of the proposed method has been evaluated by the analysis of certified reference materials. The values found presented no statistically significant differences compared to the certified values (Student`s t-test, p<0.05). In general, the relative standard deviation was lower than 12% (n = 5). (C) 2009 Elsevier B.V. All rights reserved.

In situ generation of n-butanethiol and its reaction with electron-deficient olefines

n-Butanethiol is generated in situ by sequential addition of n-butyllithium and water to elemental sulfur. The n-butanethiol formed was reacted with electron-deficient olefines to give Michael-type addition products in good yields. The method avoids the manipulation of the bad-smelling n-butanethiol.

Effect of Calcium Pre-rinse and Fluoride Dentifrice on Enamel and on Dental Plaque Formed In Situ

Purpose: The aim of this in situ double-blind randomised crossover study was to investigate the effect of calcium (Ca) pre-rinse on the composition of plaque and on enamel prior to the use of fluoride (F) dentifrice. Materials and Methods: During four phases (14 days each) of this study, 10 volunteers had agreed to wear dental appliances containing two healthy bovine enamel blocks. A fresh solution containing 20% weight/volume (w/v) sucrose was dripped on the enamel blocks ex vivo for 5 min three times a day. Subsequently, the appliances were replaced in the mouth, and the volunteers rinsed their mouth with 10 mL of a Ca (150 mmol/L) or a placebo rinse (1 min). In sequence, a slurry (1:3 w/v) of F (1030 ppm) or placebo dentifrice was dripped onto the blocks ex vivo for 1 min. During this time, the volunteers brushed their teeth with the respective dentifrice. The appliances were replaced in the mouth, and the volunteers rinsed their mouth with water. The plaque formed on the blocks was analysed for F and Ca. The enamel demineralisation as well as the incorporation of F on enamel was evaluated by cross-sectional microhardness and alkali-soluble F analysis, respectively. Data were tested using analysis of variance (P < 0.05). Results: The Ca pre-rinse prior to the use of the F dentifrice led to a three- and sixfold increase in the plaque F and Ca concentrations, respectively. It also did not have any additive effect on the F content on the enamel and the demineralisation of the enamel, in comparison with the use of F dentifrice alone. Conclusions: A Ca lactate rinse used prior to the F dentifrice was able to change the mineral content in the plaque, but it was unable to prevent enamel demineralisation.

PtSnCe/C electrocatalysts for ethanol oxidation: DEFC and FTIR ""in-situ"" studies

The ethanol oxidation reaction (EOR) was investigated using PtSnCe/C electrocatalysts in different mass ratios (72:23:5, 68:22:10 and 64:21:15) that were prepared by the polymeric precursor method. Transmission electron microscopy (TEM) showed that the particles ranged in size from approximately 2 to 5 nm. Changes in the net parameters observed for Pt suggest the incorporation of Sn and Ce into the Pt crystalline network with the formation of an alloy between Pt, Sn and/or Ce. Among the PtSnCe catalysts investigated, the 68:22:10 composition showed the highest activity toward ethanol oxidation, and the current time curves obtained in the presence of ethanol in acidic media showed a current density 50% higher than that observed for commercial PtSn/C (E-Tek). During the experiments performed on single direct ethanol fuel cells, the power density for the PtSnCe/C 68:22:10 anode was nearly 40% higher than the one obtained using the commercial catalyst. Data from Fourier transform infrared (FTIR) spectroscopy showed that the observed behavior for ethanol oxidation may be explained in terms of a double mechanism. The presence of Sn and Ce seems to favor CO oxidation, since they produce an oxygen-containing species to oxidize acetaldehyde to acetic acid. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

In Situ Infrared (FTIR) Study of the Mechanism of the Borohydride Oxidation Reaction on Smooth Pt Electrode

Although Pt has been thoroughly studied regarding its activity for the borohydride oxidation reaction (BOR), the BOR mechanism at Pt remains unclear: Depending on the applied potential, spontaneous BH(4)(-) hydrolysis can compete with the direct BOR. The goal of the present work is to provide more insight into the behavior of smooth Pt electrodes toward the BOR, by coupling in situ infrared reflectance spectroscopy with electrochemistry. The measurements were performed on a Pt electrode in 1 M NaOH/1 M NaBH(4), so as to detect the reaction intermediate species generated as a function of the applied potential. Several bands were monitored in the B-H ((v) over bar approximate to 1180, 1080, and 972 cm(-1)) and B-O ((v) over bar = 1325 and similar to 1425 cm(-1)) bond regions upon increased electrode polarization. These absorption bands, which appear sequentially and were already detected for similar measurements on Au electrodes, are assigned to BH(3), BH(2), and BO(2)(-) species. In light of these experimental data and previous results obtained in our group for Pt- or Au-based electrodes, possible initial elementary steps of the BOR on platinum electrodes are proposed and discussed according to the relevant literature data.