Morphological Analysis of the Interaction of Charged Surfactant Vesicles (SVs) with Human Cultured Cells

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.     

Endo‐ and exo‐inulinases: Enzyme‐substrate interaction and rational immobilization

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

HOCTAR database: a unique resource for microRNA target prediction

An Antarctic strain (NJ-7) of Chlorella vulgaris possesses the same 18S rRNA sequence as that of a temperate strain (UTEX259), but shows significantly higher freezing tolerance than the latter. Suppression subtractive hybridization (SSH) was performed to identify genes of intensified expression in NJ-7 relative to UTEX259. Among the genes identified, Ccor1 and Ccor2, co-organized in the same gene cluster Ccor1-Ccor2-Ccor1-Ccor2, showed much higher expression levels in NJ-7 than in UTEX259 at both 20°C and 4°C. As detected by Northern blot and Western blot analyses, the two genes were cold-inducible in NJ-7 but almost not expressed in UTEX259. Their encoded products are predicted to share 55.7% identity to each other and possess physicochemical characteristics similar to that of late embryogenesis abundant (LEA) proteins in plants. The purified recombinant Ccor1 and Ccor2 showed high heat-stability and could act as cryoprotectants to lactate dehydrogenase in vitro. Based on their expression patterns and protein characteristics, we propose that Ccor1 and Ccor2 are two novel LEA proteins and are related to the greatly enhanced freezing tolerance in the Antarctic strain.     

Heavy metals toxicity: effect of cadmium ions on amyloid beta protein 1–42. Possible implications for Alzheimer’s disease

Cadmium (Cd) is an environmental contaminant, highly toxic to humans. This biologically non-essential element accumulates in the body, especially in the kidney, liver, lung and brain and can induce several toxic effects, depending on the concentration and the exposure time. Cd has been linked to Alzheimer’s disease (AD) as a probable risk factor, as it shows higher concentrations in brain tissues of AD patients than in healthy people, its implication in the formation of neurofibrillary tangles and in the aggregation process of amyloid beta peptides (AβPs). AβPs seem to have toxic properties, particularly in their aggregated state; insoluble AβP forms, such as small and large aggregates, protofibrils and fibrils, appear to be implicated in the pathogenesis of AD. In our study, we have evaluated the effect of Cd, at different concentrations, both on the AβP1–42 ion channel incorporated in a planar lipid membrane made up of phosphatidylcholine containing 30 % cholesterol and on the secondary structure of AβP1–42 in aqueous environment. Cadmium is able to interact with the AβP1–42 peptide by acting on the channel incorporated into the membrane as well as on the peptide in solution, both decreasing AβP1–42 channel frequency and in solution forming large and amorphous aggregates prone to precipitate. These experimental observations suggesting a toxic role for Cd strengthen the hypothesis that Cd may interact directly with AβPs and may be a risk factor in AD.     

Phosphoinositide-specific Phospholipase C β1 gene deletion in bipolar disorder affected patient

The involvement of phosphoinositides (PI) signal transduction pathway and related molecules, such as the Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes, in the pathophysiology of mood disorders is corroborated by a number of recent evidences. Our previous works identified the deletion of PLCB1 gene, which codifies for the PI-PLC β1 enzyme, in 4 out 15 patients affected with schizophrenia, and no deletion both in major depression affected patients and in normal controls. By using interphase fluorescent in situ hybridization methodology, we analyzed PLCB1 in paraffin embedded samples of orbito-frontal cortex of 15 patients affected with bipolar disorder. Deletion of PLCB1 was identified in one female patient.