Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes

To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.     

Metabolic flux-based modeling of mAb production during batch and fed-batch operations

This paper proposes mathematical models that predict the physiology, growth behavior and productivity of hybridoma cells in both batch and fed-batch systems. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was employed as a model system. A systematic approach based on metabolic flux analysis (MFA) was utilized to yield a dynamic model for predicting the concentration of significant metabolites over time. Correlation analysis was performed to formulate a Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations. The coefficients of the model equation were estimated by employing the Metropolis–Hastings algorithm. The Metabolites Model was combined with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input. The prediction accuracy of the model was tested by using experimental data.     

Differential Expression and Functionality of TRPA1 Protein Genetic Variants in Conditions of Thermal Stimulation

The role of genetic modifications of the TRPA1 receptor has been well documented in inflammatory and neuropathic pain. We recently reported that the E179K variant of TRPA1 appears to be crucial for the generation of paradoxical heat sensation in pain patients. Here, we describe the consequences of the single amino acid exchange at position 179 in the ankyrin repeat 4 of human TRPA1. TRPA1 wild type Lys-179 protein expressed in HEK cells exhibited intact biochemical properties, inclusive trafficking into the plasma membrane, formation of large protein complexes, and the ability to be activated by cold. Additionally, a strong increase of Lys-179 protein expression was observed in cold (4 °C) and heat (49 °C)-treated cells. In contrast, HEK cells expressing the variant Lys-179 TRPA1 failed to get activated by cold possibly due to the loss of ability to interact with other proteins or other TRPA1 monomers during oligomerization. In conclusion, the detailed understanding of TRPA1 genetic variants might provide a fruitful strategy for future development of pain treatments.     

Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic?

In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.     

IgA Anti-β2-Glycoprotein I Autoantibodies Are Associated with an Increased Risk of Thromboembolic Events in Patients with Systemic Lupus Erythematosus

Background

The clinical utility of testing for antiphospholipid antibodies (aPL) of IgA isotype remains controversial.

Methodology/Principal Findings

To address this issue, we reasoned that if IgA aPL contribute to the clinical manifestations of the antiphospholipid syndrome, then an association with thromboembolic events should manifest in patients whose only aPL is of IgA isotype. We performed a retrospective chart review of 56 patients (31 with systemic lupus erythematosus [SLE] and 25 without SLE) whose only positive aPL was IgA anti-β2-glycoprotein I (isolated IgA anti-β2GPI) and compared their clinical features with 56 individually matched control patients without any aPL. Patients with isolated IgA anti-β2GPI had a significantly increased number of thromboembolic events, as compared to controls. When patients were stratified into those with and without SLE, the association between isolated IgA anti-β2GPI and thromboembolic events persisted for patients with SLE, but was lost for those without SLE. Titers of IgA anti-β2GPI were significantly higher in SLE patients who suffered a thromboembolic event. Among patients with isolated IgA anti-β2GPI, there was an increased prevalence of diseases or morbidities involving organs of mucosal immunity (i.e., gastrointestinal system, pulmonary system, and skin).

Conclusions/Significance

The presence of isolated IgA anti-β2GPI is associated with an increased risk of thromboembolic events, especially among patients with SLE. IgA anti-β2GPI is associated with an increased prevalence of morbidities involving organs of mucosal immunity.     

Transmission Dynamics of Highly Pathogenic Avian Influenza at Lake Constance (Europe) During the Outbreak of Winter 2005–2006

Highly pathogenic avian influenza virus (HPAI) H5N1 poses a serious threat to domestic animals. Despite the large number of studies on influenza A virus in waterbirds, little is still known about the transmission dynamics, including prevalence, behavior, and spread of these viruses in the wild waterbird population. From January to April 2006, the HPAI H5N1 virus was confirmed in 82 dead wild waterbirds at the shores of Lake Constance. In this study, we present simple mathematical models to examine this outbreak and to investigate the transmission dynamics of HPAI in wild waterbirds. The population dynamics model of wintering birds was best represented by a sinusoidal function. This model was considered the most adequate to represent the susceptible compartment of the SIR model. The three transmission models predict a basic reproduction ratio (R ₀) with value of approximately 1.6, indicating a small epidemic, which ended with the migration of susceptible wild waterbirds at the end of the winter. With this study, we quantify for the first time the transmission of HPAI H5N1 virus at Lake Constance during the outbreak of winter 2005–2006. It is a step toward the improvement of the knowledge of transmission of the virus among wild waterbirds.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and development. (β-d-glucosyl)₃ Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However, the growth was not inhibited by (α-d-galactosyl)₃ Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.