Phospholipase C and sphingomyelinase activities of the Clostridium perfringens α-toxin

α-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. α-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of α-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of α-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.     

Cumulative effect of the North Atlantic Oscillation on age‐class abundance of albacore (Thunnus alalunga)

The aim of this study was to look into possible relationships between climate and the inter‐annual variability of albacore (Thunnus alalunga) catch rates by age class observed during a recreational fishery tournament at the spawning grounds of S’Estanyol (Balearic Islands, Spain) in the years 2004–2009. The mean capture per unit effort (CPUE) was significantly higher when the mean of the North Atlantic Oscillation (NAO) experienced by the albacore in winter and spring of its life history (NAO_life) was negative than when the NAO_life was positive. A statistically significant negative relationship was obtained between NAO_life and the probability of a CPUE value for an age class and year being higher than the average CPUE for all the cohorts in that age class. The results suggest that local abundance of albacore in a spawning grounds could be related to environmental factors dependent on the NAO and that there may be a cohort‐age effect. It is hypothesized that factors dependent on the NAO, such as the abundance variation of small pelagic fishes as a response to the NAO variability, could have a cumulative effect over the good biological condition (fitness) of a long‐living fish predator such as the albacore.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.     

The effect of fragment area on site‐level biodiversity

Habitat fragmentation accompanies habitat loss, and drives additional biodiversity change; but few global biodiversity models explicitly analyse the effects of both fragmentation and loss. Here we propose and test the hypothesis that, as fragment area increases, species density (the number of species in a standardised plot) will scale with an exponent given by the difference between the exponents of the species–area relationships for islands (z ~ 0.25) and in contiguous habitat (z ~ 0.15), and test whether scaling varies between land uses. We also investigate the scaling of overall abundance and rarefaction‐based richness, as some mechanisms make different predictions about how fragment area should affect them. The relevant data from the taxonomically and geographically broad PREDICTS database were used to model the three diversity measures, testing their scaling with fragment area and whether the scaling exponent varied among land uses (primary forest, secondary forest, plantation forest, cropland and pasture). In addition, the consistency of the response of species density to fragment area was tested across three well represented taxa (Magnoliopsida, Hymenoptera and ‘herptiles’). Species density and total abundance showed area‐scaling exponents of 0.07 and 0.16, respectively, and these exponents did not vary significantly among land uses; rarefaction‐based richness by contrast did not increase consistently with area. These results suggest that the area‐scaling of species density is driven by the area‐scaling of total abundance, with additive edge effects (species moving into the small fragments from the surroundings) opposing – but not fully overcoming – the effect of fragment area on overall density of individuals. The interaction between fragment area and higher taxon (plants, vertebrates and invertebrates), which remained in the rarefied richness model, indicates that mechanisms may vary among groups.     

Long-term platinum retention after treatment with cisplatin and oxaliplatin

Background  

The aim of this study was to evaluate long-term platinum retention in patients treated with cisplatin and oxaliplatin.     

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation

Summary.  Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [³H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced. Received March 15, 2001 Accepted July 8, 2002 Published online January 20, 2003 Acknowledgements This work was supported by the Grant S1-3490 from Consejo Nacional de Investigaciones Cientificas y Tecnológicas (CONICIT), Venezuela. We appreciate the secretarial assistance of Mrs. Isabel Otaegui. Carmen I. Gutiérrez is a PhD Student from Ciencias Fisiológicas, Facultad de Medicina, Universidad Central de Venezueta (UCV), Caracas, and supported by Universidad Francisco de Miranda, Coro, Falcón, Venezuela. Joseph Glykys is a Medical Student from Universidad de Carabobo, Valencia, Venezuela, and an Assistant Student of Centro de Estudios Avanzados, IVIC. Authors' address: Dr. Lucimey Lima, Laboratorio de Neuroquímica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apdo. 21827, Caracas 1020-A, Venezuela, Fax: 58-212-504-1295, E-mail: llima@cbb.ivic.ve     

Spatial Variation of Soil CO<Subscript>2</Subscript>, CH<Subscript>4</Subscript> and N<Subscript>2</Subscript>O Fluxes Across Topographical Positions in Tropical Forests of the Guiana Shield

The spatial variation of soil greenhouse gas fluxes (GHG; carbon dioxide—CO₂, methane—CH₄ and nitrous oxide—N₂O) remains poorly understood in highly complex ecosystems such as tropical forests. We used 240 individual flux measurements of these three GHGs from different soil types, at three topographical positions and in two extreme hydric conditions in the tropical forests of the Guiana Shield (French Guiana, South America) to (1) test the effect of topographical positions on GHG fluxes and (2) identify the soil characteristics driving flux variation in these nutrient-poor tropical soils. Surprisingly, none of the three GHG flux rates differed with topographical position. CO₂ effluxes covaried with soil pH, soil water content (SWC), available nitrogen and total phosphorus. The CH₄ fluxes were best explained by variation in SWC, with soils acting as a sink under drier conditions and as a source under wetter conditions. Unexpectedly, our study areas were generally sinks for N₂O and N₂O fluxes were partly explained by total phosphorus and available nitrogen concentrations. This first study describing the spatial variation of soil fluxes of the three main GHGs measured simultaneously in forests of the Guiana Shield lays the foundation for specific studies of the processes underlying the observed patterns.