The ITGAV rs3738919-C allele is associated with rheumatoid arthritis in the European Caucasian population: a family-based study

The integrin alpha(v)beta3, whose alpha(v) subunit is encoded by the ITGAV gene, plays a key role in angiogenesis. Hyperangiogenesis is involved in rheumatoid arthritis (RA) and the ITGAV gene is located in 2q31, one of the suggested RA susceptibility loci. Our aim was to test the ITGAV gene for association and linkage to RA in a family-based study from the European Caucasian population. Two single nucleotide polymorphisms were genotyped by PCR-restriction fragment length polymorphism in 100 French Caucasian RA trio families (one RA patient and both parents), 100 other French families and 265 European families available for replication. The genetic analyses for association and linkage were performed using the comparison of allelic frequencies (affected family-based controls), the transmission disequilibrium test, and the genotype relative risk.We observed a significant RA association for the C allele of rs3738919 in the first sample (affected family-based controls, RA index cases 66.5% versus controls 56.7%; P = 0.04). The second sample showed the same trend, and the third sample again showed a significant RA association. When all sets were combined, the association was confirmed (affected family-based controls, RA index cases 64.6% versus controls 58.1%; P = 0.005). The rs3738919-C allele was also linked to RA (transmission disequilibrium test, 56.5% versus 50% of transmission; P = 0.009) and the C-allele-containing genotype was more frequent in RA index cases than in controls (RA index cases 372 versus controls 339; P = 0.002, odds ratio = 1.94, 95% confidence interval = 1.3-2.9). The rs3738919-C allele of the ITGAV gene is associated with RA in the European Caucasian population, suggesting ITGAV as a new minor RA susceptibility gene.     

A Rhipicephalus (Boophilus) microplus cathepsin with dual peptidase and antimicrobial activity

The cattle tick, Rhipicephalus (Boophilus) microplus, is a haematophagous arthropod responsible for considerable losses in the livestock industry. Immunological control with vaccines is a promising alternative to replace chemical acaricides. Due to their importance in parasite physiology, cysteine endopeptidases are potential targets. In a previous study, native Vitellin Degrading Cysteine Endopeptidase (VTDCE) was successfully tested as a vaccine antigen for bovines against R. microplus. In this work, nucleotide and amino acid VTDCE sequences were obtained from cDNA databanks, based on data from Edman sequencing and mass spectrometry. Subsequently, cloning and expression, purification, immunological and biochemical characterisation of the recombinant protein were performed to determine the biological importance of VTDCE. By Western blot, polyclonal antibodies produced against recombinant VTDCE recognised native VTDCE. Interestingly, molecular analysis showed that the VTDCE sequence has similarity to antimicrobial peptides. Indeed, experimental results revealed that VTDCE has an antimicrobial activity which is independent of endopeptidase activity. We believe that this is the first known study to show that an arthropod enzyme has antimicrobial activity.     

Buffering Capacity Explains Signal Variation in Symbiotic Calcium Oscillations

Legumes form symbioses with rhizobial bacteria and arbuscular mycorrhizal fungi that aid plant nutrition. A critical component in the establishment of these symbioses is nuclear-localized calcium (Ca2+) oscillations. Different components on the nuclear envelope have been identified as being required for the generation of the Ca2+ oscillations. Among these an ion channel, Doesn''t Make Infections1, is preferentially localized on the inner nuclear envelope and a Ca2+ ATPase is localized on both the inner and outer nuclear envelopes. Doesn''t Make Infections1 is conserved across plants and has a weak but broad similarity to bacterial potassium channels. A possible role for this cation channel could be hyperpolarization of the nuclear envelope to counterbalance the charge caused by the influx of Ca2+ into the nucleus. Ca2+ channels and Ca2+ pumps are needed for the release and reuptake of Ca2+ from the internal store, which is hypothesized to be the nuclear envelope lumen and endoplasmic reticulum, but the release mechanism of Ca2+ remains to be identified and characterized. Here, we develop a mathematical model based on these components to describe the observed symbiotic Ca2+ oscillations. This model can recapitulate Ca2+ oscillations, and with the inclusion of Ca2+-binding proteins it offers a simple explanation for several previously unexplained phenomena. These include long periods of frequency variation, changes in spike shape, and the initiation and termination of oscillations. The model also predicts that an increase in buffering capacity in the nucleoplasm would cause a period of rapid oscillations. This phenomenon was observed experimentally by adding more of the inducing signal.Plant growth is frequently limited by the essential nutrients nitrogen and phosphorus. Several plant species have established symbiotic relationships with microorganisms to overcome such limitations. In addition to the symbiotic relationship with arbuscular mycorrhizal fungi that many plants establish in order to secure their uptake of water, phosphorus, and other nutrients (Harrison, 2005; Parniske, 2008), legumes have developed interactions with bacteria called rhizobia, resulting in the fixation of atmospheric nitrogen within the plant root (Lhuissier et al., 2001; Gage, 2004; Riely et al., 2006).Root symbioses initiate with signal exchanges in the soil. Plant signals are perceived by the symbionts, triggering the successive release of microbial signals. For rhizobia, the signal molecules are lipochitooligosaccharides termed Nod factors (Dénarié et al., 1996), and the release of lipochitooligosaccharides has also been found in the fungal interaction (Maillet et al., 2011). Upon receiving diffusible signals from the microsymbionts, the plant roots initiate developmental programs that lead to infection by rhizobia or arbuscular mycorrhizal fungi. Both programs employ the same signaling pathway with several components being common to both mycorrhizal and rhizobial interactions (Kistner and Parniske, 2002; Lima et al., 2009). In particular, both the symbioses show characteristic perinuclear and nucleoplasmic localized calcium (Ca2+) oscillations, so-called Ca2+ spiking (Oldroyd and Downie, 2006; Sieberer et al., 2009). It has been suggested that Ca2+ is released from an internal store, most likely the nuclear lumen and associated endoplasmic reticulum (ER; Matzke et al., 2009), with targeted release in the nuclear region (Capoen et al., 2011).Genetic screens in the model legume Medicago truncatula have identified several genes that are required for the plant in the establishment of both symbioses. Two of these, Doesn’t Make Infections1 (DMI1) and DMI2, are essential for the induction of the Ca2+ oscillations, yet the precise roles and mechanisms of these components remain to be determined. DMI2 codes for a plasma membrane receptor-like kinase (Endre et al., 2002; Stracke et al., 2002) that is required to facilitate further signal transduction in the cell (Bersoult et al., 2005). DMI1 is a cation channel located preferentially on the inner nuclear envelope (Ané et al., 2004; Edwards et al., 2007; Riely et al., 2007; Charpentier et al., 2008; Capoen et al., 2011; Venkateshwaran et al., 2012). DMI3 encodes a calcium calmodulin-dependent protein kinase that interacts with downstream components and is thought to be the decoder of the Ca2+ oscillations (Lévy et al., 2004; Mitra et al., 2004; Hayashi et al., 2010). Additional genes coding for three nucleoporins called NUP85, NUP133, and NENA are also required for Ca2+ oscillations (Kanamori et al., 2006; Saito et al., 2007; Groth et al., 2010). The nuclear pore might be involved in trafficking secondary signals and/or ion channels to the inner nuclear membrane. These shared signaling components are collectively referred to as the common Sym pathway.DMI1 plays a key role in the production of Ca2+ oscillations, but its exact mechanism is still unknown. Orthologs of DMI1 have been found; in Lotus japonicus, they are called CASTOR and POLLUX (Charpentier et al., 2008), and in pea (Pisum sativum), SYM8 (Edwards et al., 2007). CASTOR and POLLUX, as well as calcium calmodulin-dependent protein kinase, are highly conserved both in legumes and nonlegumes (Banba et al., 2008; Chen et al., 2009). This highlights the essential role of the Ca2+ oscillations in mycorrhizal signaling.DMI1 is not the channel responsible for the release of Ca2+ (Charpentier et al., 2008; Parniske, 2008; Venkateshwaran et al., 2012) but probably influences the activity of Ca2+ channels. This is similar to how some K⁺ channels act in other plants and yeast (Peiter et al., 2007). Indeed, DMI1 is possibly a K⁺-permeable channel, based on the observation that POLLUX complements K⁺ transport in yeast (Charpentier et al., 2008). In symbiosis, the mode of action of DMI1 could be to allow cations into the nuclear envelope and in that way counterbalance the transmembrane charge that would occur following the release of Ca2+ into the nucleoplasm and cytoplasm. The cation channel could thus change the electrical potential across the nuclear membranes, affecting the opening of the voltage-activated Ca2+ channels (Edwards et al., 2007). This hypothesis is supported by a study reporting a membrane potential over the nuclear envelope in plants (Matzke and Matzke, 1986).Pharmacological evidence and the characteristics of the Ca2+ oscillations supports the involvement of Ca2+ pumps and Ca2+ channels (Engstrom et al., 2002). The pumps are needed to resequester Ca2+ after each release event, actively transporting Ca2+ against the concentration gradient using ATP. A recent study found a SERCA-type Ca2+ ATPase, MCA8, that is located on the inner and outer nuclear envelope of M. truncatula and is required for the symbiotic Ca2+ oscillations (Capoen et al., 2011). Such SERCA pumps are widely distributed on plant membranes, and the variation in their structure points to them being differentially regulated (Sze et al., 2000).Ca2+ channels release Ca2+ from the store, and the mechanism of activating these Ca2+ channels has been hypothesized to be voltage gated (Edwards et al., 2007; Oldroyd and Downie, 2008), but this remains to be verified experimentally. After release of Ca2+ into the cytosol and nucleoplasm, buffers quickly bind to and remove these free ions due to their toxicity to the cell (Sanders et al., 2002). Buffers, i.e. molecules that can bind Ca2+, may play an important role in determining the nonlinear behavior of the oscillatory system for Ca2+ signaling (Falcke, 2004). As numerous Ca2+ buffers are present in cells, it is important to take their contribution into account. Such buffers can also include experimentally introduced dyes and Ca2+ chelators.In Capoen et al. (2011), we investigated the establishment and transmission of spatial waves across the nuclear envelope and demonstrated that the key components for Ca2+ spiking reside on the inner and outer surface of the nuclear membrane. The computational framework we employed for this analysis makes a number of approximations in order to provide the computational efficiency required to perform spatiotemporal simulations. Here, a main focus is to understand the effect of buffers on the Ca2+ oscillations.In this article, we propose a mathematical model based on three key proteins; a Ca2+ ATPase, a voltage-gated Ca2+ channel, and the cation channel DMI1. The model reproduces the symbiotic Ca2+ oscillations, and we further demonstrate that Ca2+-binding proteins can explain initiation, termination, and experimentally observed variation in oscillation patterns. Furthermore, the model predicts that increases in buffering capacity can cause a period of rapid oscillations, and these were observed experimentally.     

Influence of surface treatments on the flexural strength of denture base repair

doi: 10.1111/j.1741‐2358.2011.00454.x Influence of surface treatments on the flexural strength of denture base repair Objective: The purpose of this study was to evaluate the flexural strength of repairs made with autopolymerising acrylic resin after different treatments of joint surfaces. Material and Methods: Fifty rectangular specimens were made with heat‐polymerised acrylic resin and 40 were repaired with autopolymerising acrylic resin following joint surface treatments: group 1 (intact specimens), group 2 (chemical treatment: wetting with methyl‐methacrylate for 180 s), group 3 (abraded with silicon carbide paper), group 4 (abraded and wetting with methyl‐methacrylate for 180 s) and group 5 (without surface treatment). The flexural strength was measured by a three‐point bending test using a universal testing machine with a 100 Kgf load cell in the centre of repair at 5 mm/min cross‐head speed. All data were analysed using one‐way anova and Tukey HSD test for multiple comparisons (p < 0.05). Results: Among repaired specimens, groups 2 and 4 had 66.53 ± 3.4 and 69.38 ± 1.8 MPa mean values and were similar. These groups had superior flexural strength than groups 3 and 5 that were similar and had 54.11 ± 3.4 and 51.24 ± 2.8 MPa mean values, respectively. Group 1 had a mean value of 108.30 ± 2.8 MPa being the highest result. Conclusion: It can be concluded that the treatment of the joint surfaces with methyl‐methacrylate increases the flexural strength of denture base repairs, although the strength is still lower than that observed for the intact denture base resin. Abrasion with sandpaper was not able to influence the flexural strength of repaired denture bases.     

Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1

Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-( N -ethyl-β- d -ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3–100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K_M. This was mimicked by adenylate cyclase activation (forskolin, 10 μM) and prevented by protein kinase A (PKA) inhibition ( N -[2-( p -bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 μM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 μM) facilitated GABA transport whereas PKC activation (4-β-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.     

Control of allergic rhinitis and asthma test – a formal approach to the development of a measuring tool

Background

The concurrent management of allergic rhinitis and asthma (ARA) has been recommended by Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines. However, a tool capable of assessing simultaneously the control of upper and lower airways diseases is lacking.

Aim

To describe the studies conducted to design the control of ARA test (CARAT) questionnaire.

Methods

We performed a literature review to generate a list of potentially important items for the assessment of control of ARA. A formal consensus development process, that used an innovative web-based application, was designed – 111 experts in ARA and 60 patients participated. At the final consensus meeting, 25 primary and secondary care physicians formulated the questions and response options. A qualitative feasibility study (n = 31 patients) was conducted to evaluate the comprehensibility of the questionnaire while testing two different designs.

Results

Thirty-four potentially important items were identified. All the steps of the consensus process were completed in 2.5 months. The opinions of experts and patients lead to the formulation of 17 questions. At the feasibility study the instructions and wording problems were corrected and a semi-tabular format was chosen.

Conclusion

A tool to measure the control of allergic rhinitis and asthma was developed using a comprehensive set of methodological steps ensuring the design quality and the face and content validity. Additional validation studies to assess the psychometric properties of the questionnaire have started.     

Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria

Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation.