Maitotoxin-Evoked γ-Aminobutyric Acid Release Is Due Not Only to the Opening of Calcium Channels

The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of gamma-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 microM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.     

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.     

Isolation and characterization of an inter-alpha-trypsin inhibitor-IgG complex from human serum

Inter-alpha-trypsin inhibitor is a human serum protease inhibitor of Mr 180 000 which may release physiological derivatives. A complex between IgG and an inter-alpha-trypsin inhibitor derivative of Mr 30 000 has been recently detected in human serum and was found to be inactive against trypsin, in contrast with the known inhibitory activity of the free 30-kDa derivative. The present study deals with detailed characterization of an inter-alpha-trypsin inhibitor-IgG complex following its purification by affinity chromatography techniques (anti-inter-alpha-trypsin inhibitor immunoadsorbent and Protein A-Sepharose) in mild conditions. The resulting product reacted simultaneously with anti-IgG and anti-inter-alpha-trypsin inhibitor antibodies. This complex contained Mr 180 000 inhibitor at least to some extent. It migrated in the beta-gamma zone in agarose; its molecular weight was estimated to be 1 500 000 or more; part of it displayed covalent bonding between inter-alpha-trypsin inhibitor and IgG; it had a trypsin inhibitor activity. Immunoelectrophoresis allowed one to demonstrate the native complex in serum owing to the use of anti-inter-alpha-trypsin inhibitor and anti-gamma radioactively labelled antibodies. The double immunoreactivity thus evidenced proved to be heterogeneous with respect to its level and location in the native as well as in the purified complex.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

Biochemical and strain properties of CJD prions: complexity versus simplicity

Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.     

Temperature, but not productivity or geometry, predicts elevational diversity gradients in ants across spatial grains

Aim  This research aims to understand the factors that shape elevational diversity gradients and how those factors vary with spatial grain. Specifically, we test the predictions of the species–productivity hypothesis, species–temperature hypothesis, the metabolic theory of ecology and the mid-domain effects null model. We also examine how the effects of productivity and temperature on richness depend on spatial grain.
Location  Deciduous forests along an elevational gradient in Great Smoky Mountains National Park, USA.
Methods  We sampled 22 leaf litter ant assemblages at three spatial grains, from 1-m² quadrats to 50 × 50 m plots using Winkler samplers.
Results  Across spatial grains, warmer sites had more species than did cooler sites, and primary productivity did not predict ant species richness. We found some support for the predictions of the metabolic theory of ecology, but no support for the mid-domain effects null model. Thus, our data are best explained by some version of a species–temperature hypothesis.
Main conclusions  Our results suggest that temperature indirectly affects ant species diversity across spatial grains, perhaps by limiting access to resources. Warmer sites support more species because they support more individuals, thereby reducing the probability of local extinction. Many of our results from this elevational gradient agree with studies at more global scales, suggesting that some mechanisms shaping ant diversity gradients are common across scales.     

Bioenergy production from waste: examples of biomethane and biohydrogen

This new century addresses several environmental challenges among which distribution of drinking water, global warming and availability of novel renewable energy sources to substitute for fossil fuels are of utmost importance. The last two concerns are closely related because the major part of carbon dioxide (CO(2)), considered as the main cause of the greenhouse effect, is widely produced from fossil fuel combustion. Renewable energy sources fully balanced in CO(2) are therefore of special interest, especially the issue of biological production from organic wastes. Among the possibilities of bioenergy production from wastes, two approaches are particularly interesting: The first one is relatively old and related to the production of biomethane by anaerobic digestion while the second one, more recent and innovative, relies on biohydrogen production by microbial ecosystems.     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.