Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.     

Microbiological and immunological study of staphylococcus vaccine effects in periodontitis

The aim of this study was to evaluate the immunomodulatory effect of the staphylococcal vaccine inoculated subcutaneously in 15 patients with chronic periodontitis. Bacteriological investigation of samples collected from the periodontal pocket for aerobic and anaerobic microorganisms was performed by classic bacteriological procedures before and after vaccination. The following immune system parameters were evaluated: C reactive protein (CRP), serum level of C3 complement fraction, IgG, IgA, and IgM by immunodiffusion, PMN granulocytes ROS release after in vitro stimulation with opsonized zymosan (OZ) and Concanavalin A (ConA) by chemiluminescence assay and lymphocytes sets and subsets by flow-cytometry immunophenotyping. The microbiological investigations revealed high frequency of Staphylococcus spp isolation and the presence of the most common anaerobe agents incriminated in human periodontitis like Fusobacterium, Porphyromonas, Peptostreptococcus, Veillonella spp and the reduction of this flora in the periodontal pocket after therapy. The immunological parameters quantification showed the absence of CRP, normal values of C3, IgG, IgA, IgM in the majority of cases. All patients presented normal values of lymphocytes sets and subsets. Significant increase of PMN respiratory burst after ConA stimulation was observed before vaccination which turned to normal values after therapy and a low ROS level both before and after therapy suggesting PMN Fc receptors dysfunction in this group of patients. The data presented in our study suggest an immunomodulatory effect of staphylococcal vaccine therapy in periodontitis and high frequency of Staphylococcus spp recovering from the periodontal pocket of investigated subjects.     

Evidence for immune defects in breast and lung cancer patients

Immunosuppression is often identified in cancer patients. The aim of this study was to evaluate several immune parameters for patients with breast and lung cancer. Immunophenotyping analysis showed that the cancer patients investigated had significantly lower absolute numbers of peripheral blood lymphocytes than controls. The immunosuppression was more evident for the breast cancer subgroup. The most severe immune defect noticed was the marked impairment of IFN- secretion. A shift toward the Th2 phenotype as revealed by assessment of intracellular level of IFN- and IL-4 was also noticed. The secretion of proinflammatory cytokines IL-1 and TNF- in whole blood cultures was not impaired. Although the proportion of activated cells was slightly lower than in the control group, our results showed that both peripheral T lymphocytes and NK cells of cancer patients could be induced to express early activation marker CD69 after ex vivo mitogen stimulation. In conclusion, our study revealed several immune defects in cancer patients. This suggests that an appropriate immunotherapeutical approach might be used to restore compromised immune functions with beneficial effects on both antitumor and general immunity.     

A cyclic nucleotide modulated prokaryotic K+ channel

A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.     

High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer

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3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs.     

GPCR proteomics: mass spectrometric and functional analysis of histamine H1 receptor after baculovirus-driven and in vitro cell free expression

The human histamine H1 Receptor (hH1R) belongs to the family of G-protein coupled receptors (GPCRs), an attractive and proven class of drug targets in a wide range of therapeutic areas. However, due to the low amount of available purified protein and the hydrophobic nature of GPCRs, limited structural information is available on ligand-receptor interaction especially for the transmembrane (TM) domain regions where the majority of ligand-receptor interactions occur. During the last decades, proteomic techniques have increasingly become an important tool to reveal detailed information on the individual GPCR class, including post-translational modifications and characterizations of GPCRs binding pocket. Herein, we report the successful functional production and mass spectrometric characterization of the hH1R, after baculovirus-driven and in vitro cell-free expression. Using only MALDI-ToF, sequence coverage of more than 80%, including five hydrophobic TM domains was achieved. Moreover, we have identified an asparagine residue in the hH1R protein that is subject to N-linked glycosylation. This information would be valuable for drug discovery efforts by allowing us to further study H1R-ligand interactions using histaminergic ligands that covalently bind the hH1R, and eventually revealing binding sites of hH1R and other GPCRs.     

Calcium-dependent gating of MthK, a prokaryotic potassium channel

MthK is a calcium-gated, inwardly rectifying, prokaryotic potassium channel. Although little functional information is available for MthK, its high-resolution structure is used as a model for eukaryotic Ca(2+)-dependent potassium channels. Here we characterize in detail the main gating characteristics of MthK at the single-channel level with special focus on the mechanism of Ca(2+) activation. MthK has two distinct gating modes: slow gating affected mainly by Ca(2+) and fast gating affected by voltage. Millimolar Ca(2+) increases MthK open probability over 100-fold by mainly increasing the frequency of channel opening while leaving the opening durations unchanged. The Ca(2+) dose-response curve displays an unusually high Hill coefficient (n = approximately 8), suggesting strong coupling between Ca(2+) binding and channel opening. Depolarization affects both the fast gate by dramatically reducing the fast flickers, and to a lesser extent, the slow gate, by increasing MthK open probability. We were able to capture the mechanistic features of MthK with a modified MWC model.     

Thrombin generation is increased in patients with Clostridium difficile colitis – a pilot study

Background: There are only limited data in the literature on the thrombotic risk of patients with Clostridium difficile (CD) colitis, although this disease is widespread throughout the world.

Objective: The aim of this study was to explore thrombin generation in these patients – the best way to evaluate their coagulation.

Methods: A prospective observational study was conducted during 15 months on hospitalized patients with CD colitis. Thrombin generation was performed in platelet-poor plasma using a Ceveron^® alpha analyzer and was compared with a group of volunteer control subjects.

Results: Thirty-three patients and 51 control subjects were enrolled in the study. Two biomarkers – mean velocity index and peak thrombin – were significantly higher in patient group, compared to the control subjects (p?=?0.010, respectively, p?=?0.0395). This pattern of thrombin generation suggests that patients with CD colitis without septic shock have a potential thrombotic risk. The mean velocity index significantly correlated with the estimated related risk of death according to the Charlson age-comorbidity index.

Conclusions: The higher values of thrombin generation suggest that CD colitis increases the thromboembolic risk. The pattern of thrombin generation could identify patients with particularly higher thromboembolic risk. They are potential candidates for thromboprophylaxis strategies and monitorization.     

Double transgenic mice with type 1 diabetes mellitus develop somatic, metabolic and vascular disorders

The double transgenic mice (dTg) were obtained by mating: (i) transgenic mice expressing the hemagglutinin of influenza virus under the insulin promoter with (ii) transgenic mice expressing specific T lymphocytes with receptor for the immunodominant epitope of the same virus. In this study we show that dTg mice developed type 1 diabetes mellitus associated with hyperglycemia, low level of plasma insulin, glucosuria, weight loss and approximately 90% mortality (at 3 months biological age). The membrane of red blood cells was more sensitive to osmotic shock in diabetic mice, compared to non-diabetic mice, assessing systemic oxidative stress. Both vasoconstriction and vasorelaxation of the renal arteries decreased significantly in diabetic mice (compared to the control group of non-diabetic mice) related to the phenotypic change of endothelium and smooth muscle cells within the artery wall. This animal model, may be used in developing various strategies to study pancreatic beta-cell function, as well as for a better metabolic control conducting to a reduced risk of vascular complications.