Genetic variability in the species Rhizopus stolonifer, assessed by random amplified polymorphic DNA analysis

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.     

Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake

The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.     

Effects of staurosporine,U-73122, wortmannin, 4-hydroxynonenal and sodium azide upon the release of secreted β-amyloid precursor protein from human platelets in response to thrombin stimulation

In the present study, the release of secreted -amyloid precursor protein (APPs) in response to thrombin stimulation in platelets has been investigated. Incubation of platelets with thrombin produced a concentration-dependent release of APPs with a concomitant reduction in the APP remaining in the lysates. The response to thrombin was not affected by pretreatment for 15 min with the phospholipase C inhibitor U-73122, with the protein kinase C inhibitor staurosporine, or with hydrogen peroxide (which at the concentrations used affects the phosphoinositide signalling system in human platelets). In contrast, pretreatment with wortmannin and sodium azide reduced the responses to thrombin. These data would suggest that thrombin may cause the release of APPs from human platelets via an activation of a phospholipase C-independent pathway. Thrombin-stimulated APPs release was also reduced by 4-hydroxynonenal. This finding, if it is a phenomenon also found for CNS cells, could be of relevance to the pathogenesis of Alzheimer's disease, given that an accumulation of 4-hydroxynonenal is found in this disease.     

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens.     

Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion

Nearly 60% of Plasmodium falciparum proteins are still uncharacterized and their functions are unknown. In this report, we carried out the functional characterization of a 45 kDa protein (PF3D7_1459400) and showed its potential as a target for blood stage malaria vaccine development. Analysis of protein subcellular localization, native protein expression profile, and erythrocyte invasion inhibition of both clinical and laboratory parasite strains by peptide antibodies suggest a functional role of PF3D7_1459400 protein during erythrocyte invasion. Also, immunoreactivity screens using synthetic peptides of the protein showed that adults resident in malaria endemic regions in Ghana have naturally acquired plasma antibodies against PF3D7_1459400 protein. Altogether, this study presents PF3D7_1459400 protein as a potential target for the development of peptide-based vaccine for blood-stage malaria.Impact statementPlasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.     

Simultaneous fMRI and EEG during the Multi-Source Interference Task

Background

fMRI and EEG are two non-invasive functional imaging techniques within cognitive neuroscience that have complementary advantages to obtain both temporal and spatial information. The multi-source interference task (MSIT) has been shown to generate robust activations of the dorsal anterior cingulate cortex (dACC) on both a single-subject level and in group averages, in fMRI studies. We have now simultaneously acquired fMRI and EEG during a cognitive interference task.

Materials and Methods

Healthy volunteers were tested in an MRI scanner with simultaneous EEG and fMRI recordings during the MSIT.

Results

The interference condition significantly increased the reaction time in the task. The fMRI analyses revealed activation of dACC as expected, in all subjects at the individual level and in group analyses. The posterior cingulate cortex was de-activated. Simultaneous EEG showed the expected anterior distribution of the interference effect, as it was restricted to frontal sites within a time frame of 80–120 ms post response.

Conclusion

The MSIT task is a reliable task for interference evaluation. fMRI shows robust activation of dACC and by adding EEG, an interference effect can be noticed within a temporal interval of 80–120 ms after the response, as a CRN (correct response negativity). This means that EEG could add a more detailed temporal aspect to the fMRI data from an interference task, and that despite the hostile environment within an MRI scanner, EEG data could be used.