Design of molecular wires based on supramolecular structures for application in glucose biosensors

In the present work, the synthesis and the spectroelectrochemical characterization of a novel iron compound derived of tetra-2-pyridyl-1,4-pyrazine (TPPZ) with hexacyanoferrate species forming a very stable supramolecular complex in the presence of polypyrrole (PPY) matrix, is described. The hybrid material has shown excellent catalytic activity towards H(2)O(2) detection that makes it suitable for being used as redox mediator in a glucose biosensor. The hybrid FeTPPZFeCN/PPY film presents satisfactory detection limits and high sensitivity for H(2)O(2) in the presence of K(+) or Na(+) ions. For the glucose biosensor, a linear range up to 1.1 mmoll(-1) of glucose was observed with no interferences. In this case, the sensitivities obtained were 7.88 and 5.90 microAmmol(-1)lcm(-2) in phosphate buffer or NaCl solutions, respectively. The good sensitivity is related to the presence of a high-dimensional structure based on polypyridine type ligands providing an "electron antennae effect" facilitating electron tunneling between the protein and the electrode.     

The brain 5-HT4 receptor binding is down-regulated in the Flinders Sensitive Line depression model and in response to paroxetine administration

The 5-hydroxytryptamine (5-HT₄) receptor may be implicated in depression and is a new potential target for antidepressant treatment. We have investigated the brain 5-HT₄ receptor [³H]SB207145 binding in the Flinders Sensitive Line rat depression model by quantitative receptor autoradiography, and related this to 5-HT transporter ( S )-[ N -methyl-³H]citalopram binding. We also determined the regulation of 5-HT₄ receptor binding by 1, 14, and 21 days of paroxetine administration and subchronic 5-HT depletion, and compared this with changes in 5-HT_2A receptor [³H]MDL100907 binding. In the Flinders Sensitive Line, the 5-HT₄ receptor and 5-HT transporter binding were decreased in the dorsal and ventral hippocampus, and the changes in binding were directly correlated within the dorsal hippocampus. Chronic but not acute paroxetine administration caused a 16–47% down-regulation of 5-HT₄ receptor binding in all regions evaluated including the basal ganglia and hippocampus, while 5-HT depletion increased the 5-HT₄ receptor binding in the dorsal hippocampus, hypothalamus, and lateral globus pallidus. In comparison, the 5-HT_2A receptor binding was decreased in the frontal and cingulate cortices after chronic paroxetine administration, and markedly reduced in several regions after 5-HT depletion. Thus, the 5-HT₄ receptor binding was decreased in the Flinders Sensitive Line depression model and in response to chronic paroxetine administration.     

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.     

Bacterial Analysis of Breast Milk: A Tool to Differentiate Raynaud’s Phenomenon from Infectious Mastitis During Lactation

Lactational Raynaud’s syndrome may be misdiagnosed as infectious mastitis on the basis of the breast pain. The objective of this work was to elucidate if microbiological analysis of milk may contribute to the differentiation of both conditions. Ten lactating women clinically diagnosed by Spanish lactation consultants were included in the study. Of these, five suffered from mastitis and the remaining five suffered from Raynaud’s syndrome. Breast milk samples were inoculated on diverse culture media. Seventy isolates were selected and identified by 16S rDNA PCR sequencing. Parallel, PCR-DGGE and quantitative real-time PCR were used to assess the presence of bacterial DNA in the samples. Neither bacteria nor yeasts could be detected in the milk samples provided by the women suffering from Raynaud’s syndrome. In contrast, large numbers of bacteria were isolated from those with infectious lactational mastitis. Globally, the levels of bacterial DNA were significantly higher in the milk of mastitis-suffering women. Bacteriological analysis of milk can be an useful tool to facilitate the differential diagnosis between the infectious mastitis and Raynaud’s syndrome during lactation.     

S100B Secretion in Acute Brain Slices: Modulation by Extracellular Levels of Ca<Superscript>2+</Superscript> and K<Superscript>+</Superscript>

Hippocampal slices have been widely used to investigate electrophysiological and metabolic neuronal parameters, as well as parameters of astroglial activity including protein phosphorylation and glutamate uptake. S100B is an astroglial-derived protein, which extracellularly plays a neurotrophic activity during development and excitotoxic insult. Herein, we characterized S100B secretion in acute hippocampal slices exposed to different concentrations of K⁺ and Ca²⁺ in the extracellular medium. Absence of Ca²⁺ and/or low K⁺ (0.2 mM KCl) caused an increase in S100B secretion, possibly by mobilization of internal stores of Ca²⁺. In contrast, high K⁺ (30 mM KCl) or calcium channel blockers caused a decrease in S100B secretion. This study suggests that exposure of acute hippocampal slices to low- and high-K⁺ could be used as an assay to evaluate astrocyte activity by S100B secretion: positively regulated by low K⁺ (possibly involving mobilization of internal stores of Ca²⁺) and negatively regulated by high-K⁺ (likely secondary to influx of K⁺).     

Genome size variation and morphological differentiation within Ranunculus parnassifolius group (Ranunculaceae) from calcareous screes in the Northwest of Spain

Ranunculus parnassifolius is an orophilous plant distributed throughout Central and Southwestern Europe (Alps, Pyrenees and Cantabrian Mountains). Its evolutionary history and taxonomy are often complicated, having been little studied before now. The purpose of this article is to present flow cytometry measurements and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation of R. parnassifolius s.l. from calcareous screes in the Northwest of Spain. DNA ploidy level and morphometric analysis were determined for plants of R. parnassifolius s.l. using flow cytometry (112 individuals) and multivariate analysis (152 individuals). Specimens were collected in eight localities in the Northwest of the Iberian Peninsula. Different sample preservation methods (fresh, frozen, and herbarium specimens) were employed as well as the use of various buffers and internal standards, in order to test the reproducibility of DNA flow cytometry. Three ploidy levels were detected in the study area (diploid, tetraploid, and pentaploid), and mixed-cytotype populations were also found. The mean nuclear DNA content of the R. parnassifolius group ranged from 7.43 ± 0.185 to 7.63 ± 0.339 pg/2C in diploids and from 15.09 ± 0.161 to 15.85 ± 0.587 pg/2C in tetraploids. The analysis of the monoploid genome sizes (1Cx) did not reveal a clear difference among cytotypes. These results suggest low intraspecific variation, at least among the populations studied. In addition, a comparison of different DNA reference standards was conducted. A new value for the chicken genome size was used as internal reference standard (2C = 3.14 ± 0.155 pg), with similar results found using both animal and plant standards (Pisum sativum and Solanum lycopersicum). Finally, herbarium vouchers and frozen tissue were proved to be suitable for DNA ploidy level measurements. This study provided a first assessment of C values in the R. parnassifolius group using flow cytometry. The weak morphological distinction of the cytotypes and the existence of mixed-cytotype populations in the Northwest of Spain are reported here for the first time. The different distribution pattern of the two cytotypes is discussed.     

A two-year report of pollen influx into Tauber traps in Mar Chiquita coastal lagoon,Buenos Aires,Argentina

Modern pollen deposition and its relationship to the surrounding vegetation were studied at a coastal lagoon from the southeast of Buenos Aires Province (Argentina). Tauber traps were monitored monthly over a 2-year period in a coastal dune barrier, salt marsh and continental freshwater lake. Pollen deposition exhibited seasonal patterns with maximum values during summer and a spatial variability of increasing deposition from the coast to inland sites. The pollen spectra suggest that airborne pollen originates mainly from local vegetation with scarce representation of extraregional sources. Herbaceous pollen predominates, comprising up to 90% of the total amount with Poaceae, Chenopodiineae and Asteroideae as the main types. Hydrophytic, psammophytic and extraregional types had little influence on the pollen spectra, generally comprising <5% of the total pollen. Pollen influx–vegetation abundance discrepancies were explained considering pollination syndrome, spatial distribution and structure of vegetation.     

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C‐terminal pentapeptide

Plasmodium falciparum FK506‐binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35‐TPR and demonstrate its interaction with Hsp90 C‐terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy‐based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35‐TPR and Hsp90.     

Models of spatially restricted biochemical reaction systems

Many reactions within the cell occur only in specific intracellular regions. Such local reaction networks give rise to microdomains of activated signaling components. The dynamics of microdomains can be visualized by live cell imaging. Computational models using partial differential equations provide mechanistic insights into the interacting factors that control microdomain dynamics. The mathematical models show that, for membrane-initiated signaling, the ratio of the surface area of the plasma membrane to the volume of the cytoplasm, the topology of the signaling network, the negative regulators, and kinetic properties of key components together define microdomain dynamics. Thus, patterns of locally restricted signaling reaction systems can be considered an emergent property of the cell.