Identifying the minimal copper- and zinc-binding site sequence in amyloid-beta peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.     

Angioimmunoblastic lymphoma in a 73-year-old woman

We describe the case of a 73-year-old woman with constitutional disorder and pain in the lower limbs, leading to initial suspicion of multiple myeloma. During the diagnostic process, there were discrepancies between the clinical course and findings of complementary tests. After a fulminant clinical course for a few days, the patient died, and a postmortem diagnosis of angioimmunoblastic lymphoma was established. We review the main aspects of this highly infrequent disease, the pathogenesis of which remains uncertain.     

CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.     

A new amperometric nanostructured sensor for the analytical determination of hydrogen peroxide

A new amperometric, nanostructured sensor for the analytical determination of hydrogen peroxide is proposed. This sensor was constructed by immobilizing silver nanoparticles in a polyvinyl alcohol (PVA) film on a platinum electrode, which was performed by direct drop-casting silver nanoparticles that were capped in a PVA colloidal suspension. UV-vis spectroscopy, X-ray diffraction and transmission electron microscopy were used to give a complete characterization of the nanostructured film. Cyclic voltammetry experiments yielded evidence that silver nanoparticles facilitate hydrogen peroxide reduction, showing excellent catalytic activity. Moreover, the cronoamperometric response of modified sensors was dependent on nanoparticle lifetime. Experiments were performed, using freshly prepared solutions, after 4 and 8 days. Results concerning the quantitative analysis of hydrogen peroxide, in terms of detection limit, linear range, sensitivity and standard deviation (STD), are discussed for each tested sensor type. Utilization of two different linear ranges (40 microM to 6mM and 1.25 microM to 1.0mM) enabled the assessment of concentration intervals having up to three orders of magnitude. Moreover, the electrode made using a 4-day-old solution showed the maximal sensitivity of 128 nA microM(-1)(4090 nA microM(-1)cm(-2)), yielding a limit of detection of 1 microuM and STD of 2.5 microAmM(-1). All of these analytical parameters make the constructed sensors suitable for peroxide determination in aqueous solution.     

Headspace-SPME of <Emphasis Type="Italic">in vitro</Emphasis> shoot-cultures and micropropagated plants of <Emphasis Type="Italic">Lavandula viridis</Emphasis>

In this work the volatiles emitted from in vitro shoot-cultures and micropropagated plants of Lavandula viridis L’Hér. were characterized and compared with those obtained from the field-grown mother-plant, using headspace solid phase micro-extraction following by capillary gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS). The headspace composition consisted mainly in oxygenated monoterpenes (66.7–79.2 %), where the major constituents emitted by the mature field-grown mother-plant, in vitro shoot-cultures and micropropagated plants were 1,8-cineole (74.0, 51.9 and 57.8 %) and camphor (2.9, 15.3 and 8.7 %), respectively. The headspace of in vitro shoot-cultures and micropropagated plants showed greater amount of α-pinene, camphene, β-pinene, β-selinene and selina-3,7(11)-diene, when compared with the field-grown mother-plant.     

Thermodynamic analysis of 5' and 3' single- and 3' double-nucleotide overhangs neighboring wobble terminal base pairs

Thermodynamic parameters are reported for duplex formation of 40 self-complementary RNA duplexes containing wobble terminal base pairs with all possible 3′ single and double-nucleotide overhangs, mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest neighbor analysis, the addition of a single 3′ dangling nucleotide increases the stability of duplex formation up to 1 kcal/mol in a sequence-dependent manner. The addition of a second dangling nucleotide increases the stability of duplexes closed with wobble base pairs in an idiosyncratic manner. The results allow for the development of a nearest neighbor model, which improves the predication of free energy and melting temperature for duplexes closed by wobble base pairs with 3′ single or double-nucleotide overhangs. Phylogenetic analysis of naturally occurring miRNAs was performed. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent on the orientation of the GU closing base pair rather than the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for the 5′ single terminal overhangs adjacent to wobble closing base pairs are also presented.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.     

Life in a fragment: Evolution of foraging strategies of translocated collared brown lemurs,Eulemur collaris,over an 18-year period

While the drivers of primate persistence in forest fragments have been often considered at the population level, the strategies to persist in these habitats have been little investigated at the individual or group level. Considering the rapid variation of fragment characteristics over time, longitudinal data on primates living in fragmented habitats are necessary to understand the key elements for their persistence. Since translocated animals have to cope with unfamiliar areas and face unknown fluctuations in food abundance, they offer the opportunity to study the factors contributing to successful migration between fragments. Here, we illustrated the evolution of the foraging strategies of translocated collared brown lemurs (Eulemur collaris) over an 18-year period in the Mandena Conservation Zone, south-east Madagascar. Our aim was to explore the ability of these frugivorous lemurs to adjust to recently colonized fragmented forests. Although the lemurs remained mainly frugivorous throughout the study period, over the years we identified a reduction in the consumption of leaves and exotic/pioneer plant species. These adjustments were expected in frugivorous primates living in a degraded area, but we hypothesize that they may also reflect the initial need to cope with an unfamiliar environment after the translocation. Since fragmentation is often associated with the loss of large trees and native vegetation, we suggest that the availability of exotic and/or pioneer plant species can provide an easy-to-access, nonseasonal food resource and be a key factor for persistence during the initial stage of the recolonization.