Potent in vitro anti-Trypanosoma cruzi activity of pyridine-2-thiol N-oxide metal complexes having an inhibitory effect on parasite-specific fumarate reductase

In the search for new therapeutic tools against Chagas disease (American trypanosomiasis) palladium and platinum complexes of the bioactive ligand pyridine-2-thiol N-oxide were exhaustively characterized and evaluated in vitro. Both complexes showed high in vitro growth inhibition activity (IC(50) values in the nanomolar range) against Trypanosoma cruzi, the causative agent of the disease. They were 39-115 times more active than the antitrypanosomal drug Nifurtimox. The palladium complex showed an approximately threefold enhancement of the activity compared with the parent compound. In addition, owing to their low unspecific cytotoxicity on mammalian cells, the complexes showed a highly selective antiparasite activity. To get an insight into the mechanism of action of these compounds, DNA, redox metabolism (intraparasite free-radical production) and two parasite-specific enzymes absent in the host, namely, trypanothione reductase and NADH-fumarate reductase, were evaluated as potential parasite targets. Additionally, the effect of metal coordination on the free radical scavenger capacity previously reported for the free ligand was studied. All the data strongly suggest that trypanocidal action of the complexes could mainly rely on the inhibition of the parasite-specific enzyme NADH-fumarate reductase.     

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.     

Mitochondrial DNA from osteoarthritic patients drives functional impairment of mitochondrial activity: a study on transmitochondrial cybrids

With the redefinition of osteoarthritis (OA) and the understanding that the joint behaves as an organ, OA is now considered a systemic illness with a low grade of chronic inflammation. Mitochondrial dysfunction is well documented in OA and has the capacity to alter chondrocyte and synoviocyte function. Transmitochondrial cybrids are suggested as a useful cellular model to study mitochondrial biology in vitro, as they carry different mitochondrial variants with the same nuclear background. The aim of this work was to study mitochondrial and metabolic function of cybrids with mitochondrial DNA from healthy (N) and OA donors. In this work, the authors demonstrate that cybrids from OA patients behave differently from cybrids from N donors in several mitochondrial parameters. Furthermore, OA cybrids behave similarly to OA chondrocytes. These results enhance our understanding of the role of mitochondria in the degeneration process of OA and present cybrids as a useful model to study OA pathogenesis.     

Light-induced absorption spectra of the D1–D2–cytochrome b 559 complex of Photosystem II: Effect of methyl viologen concentration

The light-induced difference absorption spectra associated to the photo-accumulation of reduced pheophytin a were studied in the isolated D1–D2–Cyt b559 complex in the presence of variable methyl viologen concentrations and different illumination conditions under anaerobiosis. Depending on the methyl viologen/reaction centre ratio, the relative intensities of the spectral bands at 681.5±0.5, 667.0±0.5 and 542.5±0.5 nm were modified. The reduced pheophytin a located at the D1-branch of the complex absorbs at 681.7±0.5 nm, and at least two additional pigment species contribute to the Q_y band of the difference absorption spectra with maxima at 667.0±0.5 and 680.5±0.5 nm. We propose the additional species correspond to a peripheral chlorophyll a and the pheophytin a located at the D2-branch of the complex, respectively. The blue absorbing chlorophyll at 667 nm is susceptible to chemical redox changes with a midpoint reduction potential of +470 mV. The Q_x absorption bands of both pheophytins localised at the D2- and D1-branch of the D1–D2–Cyt b559 complex were at 540.7±0.5 and 542.9±0.5, respectively. The results indicated that the two pheophytin molecules can be photoreduced in the D1–D2–Cyt b559 complex in certain experimental conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dermatoglyphic variation in Spanish Basque populations

The present study involves the evaluation of digital dermatoglyphic traits of 2185 unrelated individuals (1152 females and 1033 males) from 17 natural valleys of the four Basque provinces (Vizcaya, Guipúzcoa, Navarra, and Alava) in the Spanish Basque Country. Univariate intervalley and between-sex comparisons were carried out by means of chi-square contingency analysis for pattern types and by means of one-way analysis of variance for ridge counts. Multivariate intervalley comparison was carried out by means of correspondence analysis for pattern types and by principal component analysis for ridge counts. The results of this study are notable for the following findings: (1) in general, all variables are significantly heterogeneous among valley populations; (2) there was a greater differentiation among the valley populations than between sexes in one valley population; (3) affinities among the intervalley populations depend on the variables considered; (4) the valley populations from Vizcaya resemble those from the Pyrenees; (5) based on interprovince comparisons, the Vizcaya and Navarra samples are the closest: (6) in general, the valley samples from Alava are the worst clustered; (7) the universality of dermatoglyphic component structure fits better in males.     

The inhibition of semicarbazide-sensitive amine oxidase by aminohexoses

Semicarbazide-sensitive amine oxidase (EC 1.4.3.6; amine:oxygen oxidoreductase (deaminating) (copper-containing); SSAO) is a multifunctional protein. It acts under inflammatory conditions as a vascular-adhesion protein (VAP-1), mediating the adhesion of lymphocytes to vascular endothelial cells. The relationships, if any, between this adhesion function and the enzymatic functions (amine-substrate specificity and catalysis) of SSAO have not yet been defined. Since cell surface amino sugars and their derivatives are known to be involved in cell-to-cell recognition, we have investigated their possible effects on the enzyme activity of SSAO. The aminohexoses galactosamine, glucosamine and mannosamine were not oxidatively deaminated by SSAO. However, their presence during the assay of benzylamine oxidation resulted in a time-dependent inhibition. This inhibition was shown to follow saturation kinetics with respect to hexosamine concentration. Although time-dependent, the inhibition of SSAO activity was found to be reversible by dilution. In contrast, there is no such inhibition when the N-acetylamino sugar derivatives or the parent sugars (galactose, glucose and mannose) replaced the amino sugars in the reaction mixture. These results suggest that the interactions between SSAO and aminohexoses are specific and, therefore, that the cell-adhesion functions and amine-recognition functions of VAP-1/SSAO may be interlinked.     

Semicarbazide-sensitive amine oxidases in pig dental pulp

The behaviour of semicarbazide-sensitive amine oxidase (SSAO; E.C. 1.4.3.6) in dental pulp has been studied, with particular reference to the metabolism of 5-hydroxytryptamine (5-HT; serotonin). Kinetic studies using radioactively labelled substrates have confirmed benzylamine, 2-phenylethylamine (PEA) and 5-HT to be substrates for microsomal SSAO from porcine dental pulp. Kinetic substrate-competition studies indicated the presence of two forms of SSAO in dental pulp; one that oxidises benzylamine and PEA but not 5-HT and a second that oxidises 5-HT and PEA but not benzylamine. These two forms also differ in their thermostabilities at 60 and 70 degrees C, although this thermal inactivation is partly reversible.     

Molecular study of a new family with hereditary renal cell carcinoma and a translocation t(3;8)(p13;q24.1)

An alternative model has been proposed for the development of clear-cell renal cell carcinoma (RCC) in families where chromosome 3 translocations segregate with the disease. In this model, the existence of a translocation involving chromosome 3 would favour the non-disjunctional loss of the derivative chromosome carrying the 3p segment. Additionally, subsequent somatic mutations in the VLH gene, located in 3p25-26, would inactivate this tumour suppressor gene. In the present work, we describe a new family with two clear-cell RCC affected members and a t(3;8)(p13;q24.1) translocation in two consecutive generations. We observed loss of the derivative chromosome carrying the 3p segment (der(8)) and somatic mutation of the VHL gene in the left-kidney tumoral tissue of the proband. His right-kidney tumour carried a different VHL mutation and loss of heterozygosity (LOH) was not detected. The mother of the proband was also clear-cell RCC-affected but the tumoral tissue analysed did not carry any VHL gene mutations. Another member of the family, a maternal aunt, had a papillary RCC and did not carry this translocation, the LOH on 3p or the VHL somatic mutations. Haplotype analysis of the three affected members revealed that they did not inherit a common region on 3p, confirming the different genetic origin of both tumour types. Finally, the presence of RCC in other non-available members of the family highlights the overall risk for RCC in families with chromosome 3 translocations. In the present work, we have confirmed the proposed mechanism for the development of clear-cell RCC in this family, although we cannot discard the existence of other genes, in addition to VHL, being involved in hereditary RCC.     

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.     

Use of a diffusion model for mono- and bicomponent anion-exchange of two isoenzymes of glucoamylase from Aspergillus niger in a fixed bed

The adsorption of glucoamylases I and II (GA I and GA II, respectively) from Aspergillus niger on the anion exchanger DEAE-Toyopearl 650 was studied in fixed-bed experiments, and the effect of temperature, flowrate, inlet concentration, bed length, and particle size on the process was characterized. The anion exchanger showed a higher adsorption capacity for the more active isoenzyme GA I in all experimental conditions studied. A mathematical model accounting for external and pore diffusion and nonlinear equilibrium isotherm (for GA I) was used to fit the experimental breakthrough curves, showing very accurate fittings in all of the operating conditions. The values of the pore diffusion coefficient at 15, 20, and 25 degrees C were, respectively, 1.25 x 10(-)(11), 1.46 x 10(-)(11) and 1.83 x 10(-)(11) (for GA I) and 1.82 x 10(-)(11), 2.44 x 10(-)(11) and 2.73 x 10(-)(11) (for GA II) m(2)/s. Bicomponent adsorption experiments showed no significant interference effects between GA I and GA II, and so the mathematical model was again used to fit these experiments, yielding very satisfactory results.