Two splice variants derived from a Drosophila melanogaster candidate ClC gene generate ClC-2-type Cl- channels

Members of the ClC family of membrane proteins have been found in a variety of species and they can function as Cl- channels or Cl-/H+ antiporters. Three potential ClC genes are present in the Drosophila melanogaster genome. Only one of them shows homology with a branch of the mammalian ClC genes that encode plasma membrane Cl- channels. The remaining two are close to mammalian homologues coding for intracellular ClC proteins. Using RT-PCR we have identified two splice variants showing highest homology (41% residue identity) to the mammalian ClC-2 chloride channel. One splice variant (DmClC-2S) is expressed in the fly head and body and an additional, larger variant (DmClC-2L) is only present in the head. Both putative Drosophila channels conserve key features of the ClC channels cloned so far, including residues conforming the selectivity filter and C-terminus CBS domains. The splice variants differ in a stretch of 127 aa at the intracellular C-terminal portion separating cystathionate beta synthase (CBS) domains. Expression of either Drosophila ClC-2 variant in HEK-293 cells generated inwardly rectifying Cl- currents with similar activation and deactivation characteristics. There was great similarity in functional characteristics between DmClC-2 variants and their mammalian counterpart, save for slower opening kinetics and faster closing rate. As CBS domains are believed to be sites of regulation of channel gating and trafficking, it is suggested that the extra amino acids present between CBS domains in DmClC-2L might endow the channel with a differential response to signals present in the fly cells where it is expressed.     

Ecological risk assessment in a tropical wetland contaminated with gasoline: Tier 1

To assess natural attenuation and the efficiency of remediation actions after more than two years a large accident with gasoline spill contaminated a wetland in a tropical region, an Ecological Risk Assessment based on the Dutch Triad was applied. In total, eight surface water-sampling points were distributed randomly in the affected area and on reference area upstream the contaminated site, with similar ecological characteristics. Risks based on chemical, ecotoxicological, and ecological lines of evidence (LoE) were calculated to integrate the environmental risk indexes. The chemical risk was derived from toxic pressure coefficients based on the total BTEX and naphthalene concentrations. Ecotoxicological LoE based on acute toxicity with Daphnia similis and Aliivibrio fischeri bioassays and chronic toxicity with Desmodesmus subspicatus bioassay contributed to raise uncertainty due to low sensitivity of acute assays. Genotoxicity and endocrine disruption biomarkers of Oreochromis niloticus were used to calculate the Biomarker Stress Index (BSI) assumed as Ecological Risk Index. The integration of the Chemical Risk Index with BSI to estimate the Biological Vulnerability Index allowed a meaningful analysis of the threats to the aquatic ecosystem, thereby supporting managers and decision-makers.     

Double trouble—Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments

The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant “ripple effect” on subsequent studies.     

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17–19 d in the 3 highest dose groups and volume of distribution of 5–6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.     

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

Hydrogen peroxide (H₂O₂) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H₂O₂ sensor that allows H₂O₂ monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H₂O₂ sensor reduction–oxidation sensitive green fluorescent protein2-Tsa2ΔC_R to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H₂O₂ produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H₂O₂ levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H₂O₂ levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H₂O₂ biology in plant cells.

The establishment of a hypersensitive H₂O₂ sensor in six major compartments of the Chlamydomonas cell reveals steep intracellular H₂O₂ gradients under normal physiological conditions with limited diffusion into other compartments.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.     

Influence of plant resistance at the third trophic level: interactions between parasitoids and entomopathogenic fungi of cereal aphids

Host-plant resistance can affect herbivorous insects and their natural enemies such as parasitoids and entomopathogenic fungi. This tritrophic effect acts on interspecific interactions between the two groups of natural enemies distantly related in phylogenetic terms. The intra- and extra-host aspects of the interaction between the cereal aphid parasitoid Aphidius rhopalosiphi and the entomopathogenic fungus Erynia neoaphidis developing on the grain aphid, Sitobion avenae, on resistant and susceptible wheat (Triticum aestivum) cultivars, were studied. The competitive outcome of the intra-host interaction depended on the timing of parasitoid oviposition and fungal infection and was affected by wheat resistance. In particular, survival of the parasitoid was lower on the resistant wheat cultivar than the susceptible wheat cultivar, when the competitive outcome of the interaction was favourable for either parasitoid or fungal development. Before and after this period the influence of plant resistance was not significant. Furthermore, the extra-host interaction was not affected by the wheat cultivar, although an increase in fungal infection of S. avenae was observed when parasitoids foraged in the experimental arena with sporulating aphid cadavers compared with foraging in the absence of sporulating cadavers. Our results showed that the host plant may affect interspecific interactions between parasitoids and fungi and that these interactions depended on the timing of parasitoid oviposition and fungal infection. Received: 16 March 1998 / Accepted: 24 August 1998