Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.     

On the mechanism of gating charge movement of ClC-5, a human Cl(-)/H(+) antiporter

ClC-5 is a Cl(-)/H(+) antiporter that functions in endosomes and is important for endocytosis in the proximal tubule. The mechanism of transport coupling and voltage dependence in ClC-5 is unclear. Recently, a transport-deficient ClC-5 mutant (E268A) was shown to exhibit transient capacitive currents. Here, we studied the external and internal Cl(-) and pH dependence of the currents of E268A. Transient currents were almost completely independent of the intracellular pH. Even though the transient currents are modulated by extracellular pH, we could exclude that they are generated by proton-binding/unbinding reactions. In contrast, the charge movement showed a nontrivial dependence on external chloride, strongly supporting a model in which the movement of an intrinsic gating charge is followed by the voltage-dependent low-affinity binding of extracellular chloride ions. Mutation of the external Glu-211 (a residue implicated in the coupling of Cl(-) and proton transport) to aspartate abolished steady-state transport, but revealed transient currents that were shifted by ~150 mV to negative voltages compared to E268A. This identifies Glu(ext) as a major component of the gating charge underlying the transient currents of the electrogenic ClC-5 transporter. The molecular events underlying the transient currents of ClC-5 emerging from these results can be explained by an inward movement of the side chain of Glu(ext), followed by the binding of extracellular Cl(-) ions.     

Novel cyclohexyl-amides as potent antibacterials targeting bacterial type IIA topoisomerases

As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties.     

Novel amino-piperidines as potent antibacterials targeting bacterial type IIA topoisomerases

We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Obstructive lung diseases and inhaler treatment: results from a national public pragmatic survey

Background

The opinions held by the general population on obstructive lung disease and inhaler devices could influence asthma and chronic obstructive pulmonary disorder (COPD) management and treatment adherence.The aim of the present public pragmatic survey was to evaluate the opinions, beliefs and perceptions of Italian people with respect to respiratory diseases as well as their perspectives on the use of inhaler devices.

Methods

This survey was conducted on a group of 2,008 individuals forming a representative sample of the Italian population aged 15 years and over. It was based on personal interviews that were administered in the homes of the respondents using a structured questionnaire that took approximately 30 minutes.

Results

Awareness of obstructive lung diseases is poor. Asthma, but not COPD, was perceived as a common and increasingly prevalent disease by the majority of the interviewees. Allergy, pollution and smoking were considered to be responsible for both of these diseases. The rates at which the respondents claimed to be suffering from asthma and COPD were lower than expected (4% and 2%, respectively). Inhaled drugs were recognised as the main treatment by 65% of the respondents. The great majority of respondents attributed positive characteristics to the inhaler device (e.g., safety, reliability, effectiveness, ease of use and practicality). Compared to people who have never used inhaler devices, individuals who suffer from asthma or COPD were more confident in their use and showed a greater belief in their safety, reliability and trustworthiness. People older than 64 years showed less attention to the properties of these devices.

Conclusions

The present results highlight the need for public interventions aimed at improving awareness of obstructive lung disease and reveal various potentialities and critical issues for inhaler device usage. Switching of devices was considered feasible by most of the interviewees, as long as the choice is carefully explained by their physician.     

Lipopolysaccharide O-Chain Core Region Required for Cellular Cohesion and Compaction of In Vitro and Root Biofilms Developed by Rhizobium leguminosarum

The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.