Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: Involvement of their effects on mitochondrial energy-linked functions

The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ~32% even at the lowest concentration (65 nmol mg(-1) protein), reaching ~67% at the concentration of 130 nmol mg(-1) protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5-130 nmol mg(-1) protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5-65 nmol mg(-1) protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65 nmol mg(-1) protein. Lipid peroxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80 nmol mg(-1) protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ~50%, ~40% and ~58% with MI-J, MI-4F and MI-2,4diF (80 nmol mg(-1) protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ~22%, ~32% and ~40% (80 nmol mg(-1) protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.     

Removal of virus from boar semen spiked with porcine circovirus type 2

The virus porcine circovirus type 2 (PCV2) is associated with different disease entities, including reproductive failure. The objective of this study was to investigate the use of a semen processing technique for the elimination of infectious PCV2 in semen. PCV2 was chosen as a model virus because of its small size, high resistance to inactivation and as a known risk factor for boar semen contamination. Aliquots of ejaculates were spiked with PCV2 and processed by a double processing technique, consisting of Single Layer Centrifugation on Androcoll?-P followed by a "swim-up" procedure. Samples were collected from the resulting fractions during the selection process and analyzed for the presence of infectious PCV2. Virus titres were determined by performing a 50% tissue culture infective dose assay (TCID(50)) by end point dilution and with the use of an indirect peroxidise monolayer assay technique. With an initial infectious virus titre of 3.25-3.82 (TCID(50))/50μL the two-step sperm selection method eliminated 2.92±0.23 logs of infectious PCV2, corresponding to more than 99% reduction. Sperm quality was not affected by the selection procedure.     

Secretion of adipokines by human adipose tissue in vivo: partitioning between capillary and lymphatic transport

Peptides secreted by adipose tissue (adipokines) may enter blood via capillaries or lymph. The relative importance of these pathways for a given adipokine might influence its biological effects. Because this has not been studied in any species, we measured the concentrations of seven adipokines and eight nonsecreted proteins in afferent peripheral lymph and venous plasma from 12 healthy men. Data for nonsecreted proteins were used to derive indices of microvascular permeability, which in conjunction with the molecular radii of the adipokines were used to estimate the amounts leaving the tissue via capillaries. Transport rates via lymph were estimated from the lymph adipokine concentrations and lymph flow rates and total transport (secretion) as the sum of this and capillary transport. Concentrations of nonsecreted proteins were always lower in lymph than in plasma. With the exception of adiponectin, adipokine concentrations were always higher in lymph (P < 0.01). Leptin and MCP-1 were secreted at the highest rates (means: 43 μg/h or 2.7 nmol/h and 32 μg/h or 2.4 nmol/h, respectively). IL-6 and MCP-1 secretion rates varied greatly between subjects. The proportion of an adipokine transported via lymph was directly related to its molecular radius (r(s) = +0.94, P = 0.025, n = 6), increasing from 14 to 100% as the radius increased from 1.18 (IL-8) to 3.24 nm (TNFα). We conclude that the lymph/capillary partitioning of adipokines is a function of molecular size, which may affect both their regional and systemic effects in vivo. This finding may have implications for the physiology of peptides secreted by other tissues.     

Genetic diversity in clinical isolates of Mycobacterium avium complex from Guinea-Bissau, West Africa

Isolates of Mycobacterium avium complex (MAC) were cultured from sputum samples obtained from patients in Guinea-Bissau, West Africa. Twenty-eight isolates hybridising with MAC probe (AccuProbe) were further characterised by different molecular techniques: hybridisation with species-specific probes (AccuProbe) for M. avium and M. intracellulare, partial sequencing of 16S rRNA gene and PCR detection of the DT1-DT6 sequences and the macrophage-induced gene (mig). Only one of the 28 isolates reacted with the M. avium probe and four with the M. intracellulare probe. Two isolates expressed the DT1 sequence, and three the DT6. The mig was detected in 18 (64%) of the isolates. Sequencing of 16S rRNA had the greatest discriminative power of the typing methods applied, without strong correlation with any other technique. Clinical MAC isolates from Guinea-Bissau demonstrated a wide genetic diversity among the members of M. avium complex that might reflect on biotope variation.     

The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus

The ability to elicit broadly neutralizing antibody responses against HIV-1 is a crucial goal for a prophylactic HIV-1 vaccine. Here, we discuss the difficulties of achieving broad HIV-1 neutralization in the context of both the effective annual human influenza virus vaccine and the need to develop a pandemic influenza vaccine. Immunogen-design strategies are underway to target functionally conserved regions of the HIV-1 envelope glycoproteins, and similar strategies might be applicable to pandemic influenza virus vaccine development. Efforts to develop broadly neutralizing vaccines against either HIV-1 or influenza virus might establish a paradigm for future vaccines against highly variable pathogens.     

Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator‐dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.     

Lipoprotein size is a main determinant for the rate of hydrolysis by exogenous LPL in human plasma

LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietin-like proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.     

The response of soil solution chemistry in European forests to decreasing acid deposition

Acid deposition arising from sulphur (S) and nitrogen (N) emissions from fossil fuel combustion and agriculture has contributed to the acidification of terrestrial ecosystems in many regions globally. However, in Europe and North America, S deposition has greatly decreased in recent decades due to emissions controls. In this study, we assessed the response of soil solution chemistry in mineral horizons of European forests to these changes. Trends in pH, acid neutralizing capacity (ANC), major ions, total aluminium (Al_tot) and dissolved organic carbon were determined for the period 1995–2012. Plots with at least 10 years of observations from the ICP Forests monitoring network were used. Trends were assessed for the upper mineral soil (10–20 cm, 104 plots) and subsoil (40–80 cm, 162 plots). There was a large decrease in the concentration of sulphate () in soil solution; over a 10‐year period (2000–2010), decreased by 52% at 10–20 cm and 40% at 40–80 cm. Nitrate was unchanged at 10–20 cm but decreased at 40–80 cm. The decrease in acid anions was accompanied by a large and significant decrease in the concentration of the nutrient base cations: calcium, magnesium and potassium (Bc = Ca²⁺ + Mg²⁺ + K⁺) and Al_tot over the entire dataset. The response of soil solution acidity was nonuniform. At 10–20 cm, ANC increased in acid‐sensitive soils (base saturation ≤10%) indicating a recovery, but ANC decreased in soils with base saturation >10%. At 40–80 cm, ANC remained unchanged in acid‐sensitive soils (base saturation ≤20%,  ≤ 4.5) and decreased in better‐buffered soils (base saturation >20%,  > 4.5). In addition, the molar ratio of Bc to Al_tot either did not change or decreased. The results suggest a long‐time lag between emission abatement and changes in soil solution acidity and underline the importance of long‐term monitoring in evaluating ecosystem response to decreases in deposition.     

Bacterial influence on partitioning rate during the biodegradation of styrene in a biphasic aqueous-organic system

The degradation by a consortium of slightly-halophile marine bacteria of styrene initially dissolved in silicone oil was monitored in batch reactors stirred at 75, 125 and 500 rpm, respectively. In the 75 and 125 rpm cases, the styrene biodegradation rate was higher than the rate of spontaneous partitioning of styrene from the oil to the water, determined under abiotic conditions. Abiotic transfer tests carried out after biodegradation runs revealed that bacterial activity had resulted in a significant increase in the rate of styrene partitioning between the two liquid phases. Even though bacterial adsorption was noticeable at the oil-water interface, this effect appeared to be due to the release by the bacteria of chemicals in the aqueous phase. Similarity with observations made with Triton X-100 suggested that the chemicals released may have been biosurfactants or solubilizing agents.