Receptor crosstalk: haloperidol treatment enhances A2A adenosine receptor functioning in a transfected cell model

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.     

Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies

Background  

All polypeptide backbones have the potential to form amyloid fibrils, which are associated with a number of degenerative disorders. However, the likelihood that amyloidosis would actually occur under physiological conditions depends largely on the amino acid composition of a protein. We explore using a naive Bayesian classifier and a weighted decision tree for predicting the amyloidogenicity of immunoglobulin sequences.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Proteomics and transcriptomics investigation on longissimus muscles in Large White and Casertana pig breeds

Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fat-depositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed.     

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.     

The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities

Humans contain many HSP (heat-shock protein) 70/HSPA- and HSP40/DNAJ-encoding genes and most of the corresponding proteins are localized in the cytosol. To test for possible functional differences and/or substrate specificity, we assessed the effect of overexpression of each of these HSPs on refolding of heat-denatured luciferase and on the suppression of aggregation of a non-foldable polyQ (polyglutamine)-expanded Huntingtin fragment. Overexpressed chaperones that suppressed polyQ aggregation were found not to be able to stimulate luciferase refolding. Inversely, chaperones that supported luciferase refolding were poor suppressors of polyQ aggregation. This was not related to client specificity itself, as the polyQ aggregation inhibitors often also suppressed heat-induced aggregation of luciferase. Surprisingly, the exclusively heat-inducible HSPA6 lacks both luciferase refolding and polyQ aggregation-suppressing activities. Furthermore, whereas overexpression of HSPA1A protected cells from heat-induced cell death, overexpression of HSPA6 did not. Inversely, siRNA (small interfering RNA)-mediated blocking of HSPA6 did not impair the development of heat-induced thermotolerance. Yet, HSPA6 has a functional substrate-binding domain and possesses intrinsic ATPase activity that is as high as that of the canonical HSPA1A when stimulated by J-proteins. In vitro data suggest that this may be relevant to substrate specificity, as purified HSPA6 could not chaperone heat-unfolded luciferase but was able to assist in reactivation of heat-unfolded p53. So, even within the highly sequence-conserved HSPA family, functional differentiation is larger than expected, with HSPA6 being an extreme example that may have evolved to maintain specific critical functions under conditions of severe stress.     

Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-β did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.     

A GC-MS metabolic profiling study of plasma samples from mice on low- and high-fat diets

Metabolic profiling of biofluids, based on the quantitative analysis of the concentration profile of their free low molecular mass metabolites, has been playing increasing role employed as a means to gain understanding of the progression of metabolic disorders, including obesity. Chromatographic methods coupled with mass spectrometry have been established as a strategy for metabolic profiling. Among these, GC-MS, targeting mainly the primary metabolism intermediates, offers high sensitivity, good peak resolution and extensive databases. However, the derivatization step required for many involatile metabolites necessitates specific data validation, normalization and analysis protocols to ensure accurate and reproducible performance. In this study, the GC-MS metabolic profiles of plasma samples from mice maintained on 12- or 15-month long low (10 kcal%) or high (60 kcal%) fat diets were obtained. The profiles of the trimethylsilyl(TMS)-methoxime(MeOx) derivatives of the free polar metabolites were acquired through GC-(ion trap)MS, using [U-(13)C]-glucose as the internal standard. After the application of a recently developed data correction and normalization/filtering protocol for GC-MS metabolomic datasets, the profiles of 48 out of the 77 detected metabolites were used in multivariate statistical analysis. Data mining suggested a decrease in the activity of the energy metabolism with age. In addition, the metabolic profiles indicated the presence of subpopulations with different physiology within the high- and low-fat diet mice, which correlated well with the difference in body weight among the animals and current knowledge about hyperglycemic conditions.     

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.     

Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysms

PurposeTo test the hypothesis that ruptured abdominal aortic aneurysms (AAA) are globally weaker than unruptured ones.MethodsFour ruptured and seven unruptured AAA specimens were harvested whole from fresh cadavers during autopsies performed over an 18-month period. Multiple regionally distributed longitudinally oriented rectangular strips were cut from each AAA specimen for a total of 77 specimen strips. Strips were subjected to uniaxial extension until failure. Sections from approximately the strongest and weakest specimen strips were studied histologically and histochemically. From the load-extension data, failure tension, failure stress and failure strain were calculated. Rupture site characteristics such as location, arc length of rupture and orientation of rupture were also documented.ResultsThe failure tension, a measure of the tissue mechanical caliber was remarkably similar between ruptured and unruptured AAA (group mean±standard deviation of within-subject means: 11.2±2.3 versus 11.6±3.6 N/cm; p=0.866 by mixed model ANOVA). In post-hoc analysis, there was little difference between the groups in other measures of tissue mechanical caliber as well such as failure stress (95±28 versus 98±23 N/cm²; p=0.870), failure strain (0.39±0.09 versus 0.36±0.09; p=0.705), wall thickness (1.7±0.4 versus 1.5±0.4 mm; p=0.470) , and % coverage of collagen within tissue cross section (49.6±12.9% versus 60.8±9.6%; p=0.133). In the four ruptured AAA, primary rupture sites were on the lateral quadrants (two on left; one on left-posterior; one on right). Remarkably, all rupture lines had a longitudinal orientation and ranged from 1 to 6 cm in length.ConclusionThe findings are not consistent with the hypothesis that ruptured aortic aneurysms are globally weaker than unruptured ones.