Specificity of antinuclear autoantibodies in diseases with systemic destruction of connective tissue]

By using the fluorescent antibody technique several patterns of antinuclear antibodies (ANA) were detected in the systemic connective tissue diseases, as well as in some other diseases and in healthy donors, depending on the character of nuclear fluorescence. Homogeneous nuclear fluorescence, homogeneous fluorescence free of nucleaolar fluorescence, ring-shaped fluorescence, lumpy fluorescence, selective nuclealar fluorescence, nuclear fluorescence in the form of long fine plexiform bands associated with fluorescence in the nuclear membrane region. The latter ANA pattern was found only in several forms of lupus erythematosus, and this fluorescence was different from the previously reported "reticular" and "filamentous" patterns.     

Membrane-Localized Extra-Large G Proteins and Gβγ of the Heterotrimeric G Proteins Form Functional Complexes Engaged in Plant Immunity in Arabidopsis

In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.Heterotrimeric GTP-binding proteins (G proteins), classically consisting of Gα, Gβ, and Gγ subunits, are essential signal transduction elements in most eukaryotes. In animals and fungi, ligand perception by G protein-coupled receptors leads to replacement of GDP with GTP in Gα, triggering activation of the heterotrimer (Li et al., 2007; Oldham and Hamm, 2008). Upon activation, GTP-bound Gα and Gβγ are released and interact with downstream effectors, thereby transmitting signals to multiple intracellular signaling cascades. Signaling terminates when the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP and the inactive heterotrimer reforms at the receptor. The large diversity of mammalian Gα subunits confers specificity to the multiple signaling pathways mediated by G proteins (Wettschureck and Offermanns, 2005). Five distinct classes of Gα have been described in animals (Gαi, Gαq, Gαs, Gα12 and Gαv), with orthologs found in evolutionarily primitive organisms such as sponges (Oka et al., 2009). Humans possess four classes of Gα involving 23 functional isoforms encoded by 16 genes (McCudden et al., 2005), while only a single prototypical Gα is usually found per plant genome (Urano et al., 2013). Multiple copies of Gα are present in some species with recently duplicated genomes, such as soybean (Glycine max) with four Gα genes (Blanc and Wolfe, 2004; Bisht et al., 2011). In the model plant Arabidopsis (Arabidopsis thaliana), a prototypical Gα subunit (GPA1) is involved in a number of important processes, including cell proliferation (Ullah et al., 2001), inhibition of inward K⁺ channels and activation of anion channels in guard cells by mediating the abscisic acid pathway (Wang et al., 2001; Coursol et al., 2003), blue light responses (Warpeha et al., 2006, 2007), and germination and postgermination development (Chen et al., 2006; Pandey et al., 2006).It is well established that heterotrimeric G proteins play a fundamental role in plant innate immunity. In Arabidopsis, two different Gβγ dimers (Gβγ1 and Gβγ2) are generally considered to be the predominant elements in G protein defense signaling against a variety of fungal pathogens (Llorente et al., 2005; Trusov et al., 2006, 2007, 2009; Delgado-Cerezo et al., 2012; Torres et al., 2013). By contrast, these studies attributed a small or no role to Gα, because mutants deficient in Gα displayed only slightly increased resistance against the fungal pathogens (Llorente et al., 2005; Trusov et al., 2006; Torres et al., 2013). The Gβγ-mediated signaling also contributes to defense against a model bacterial pathogen Pseudomonas syringae, by participating in programmed cell death (PCD) and inducing reactive oxygen species (ROS) production in response to at least three pathogen-associated molecular patterns (PAMPs; Ishikawa, 2009; Liu et al., 2013; Torres et al., 2013). Gα is not involved in PCD or PAMP-triggered ROS production (Liu et al., 2013; Torres et al., 2013). Nonetheless, Arabidopsis Gα plays a positive role in defense against P. syringae, probably by mediating stomatal function and hence physically restricting bacterial entry to the leaf interior (Zhang et al., 2008; Zeng and He, 2010; Lee et al., 2013). Given the small contribution from Gα, the involvement of heterotrimeric G proteins in Arabidopsis resistance could be explained in two ways: either the Gβγ dimer acts independently from Gα, raising a question of how is it activated upon a pathogen attack, or Gα is replaced by another protein for heterotrimer formation.The Arabidopsis genome contains at least three genes encoding Gα-like proteins that have been classified as extra-large G proteins (XLGs; Lee and Assmann, 1999; Ding et al., 2008). XLGs comprise two structurally distinct regions. The C-terminal region is similar to the canonical Gα, containing the conserved helical and GTPase domains, while the N-terminal region is a stretch of approximately 400 amino acids including a putative nuclear localization signal (Ding et al., 2008). GTP binding and hydrolysis were confirmed for all three XLG proteins, although their enzymatic activities are very slow and require Ca²⁺ as a cofactor, whereas canonical Gα utilizes Mg²⁺ (Heo et al., 2012). Several other features differentiate XLGs from Gα subunits. Comparative analysis of XLG1 and Gα at the DNA level showed that the genes are organized in seven and 13 exons, respectively, without common splicing sites (Lee and Assmann, 1999). XLGs have been reported to localize to the nucleus (Ding et al., 2008). Analysis of knockout mutants revealed a nuclear function for XLG2, as it physically interacts with the Related To Vernalization1 (RTV1) protein, enhancing the DNA binding activity of RTV1 to floral integrator gene promoters and resulting in flowering initiation (Heo et al., 2012). Therefore, it appears that XLGs may act independently of G protein signaling. On the other hand, functional similarities between XLGs and the Arabidopsis Gβ subunit (AGB1) were also discovered. For instance, XLG3- and Gβ-deficient mutants were similarly impaired in root gravitropic responses (Pandey et al., 2008). Knockout of all three XLG genes caused increased root length, similarly to the Gβ-deficient mutant (Ding et al., 2008). Furthermore, as observed in Gβ-deficient mutants, xlg2 mutants displayed increased susceptibility to P. syringae, indicating a role in plant defense (Zhu et al., 2009). Nevertheless, a genetic analysis of the possible functional interaction between XLGs and Gβ has not been established.In this report, we performed in-depth genetic analyses to test the functional interaction between the three XLGs and Gβγ dimers during defense-related responses in Arabidopsis. We also examined physical interaction between XLG2 and the Gβγ dimers using yeast (Saccharomyces cerevisiae) three-hybrid (Y3H) and bimolecular fluorescent complementation (BiFC) assays. Our findings indicate that XLGs function as direct partners of Gβγ dimers in plant defense signaling. To estimate relatedness of XLGs and Gα proteins, we carried out a phylogenetic analysis. Based on our findings, we conclude that plant XLG proteins most probably originated from a canonical Gα subunit and retained prototypical interaction with Gβγ dimers. They function together with Gβγ in a number of processes including plant defense, although they most probably evolved activation/deactivation mechanisms very different from those of a prototypical Gα.     

Specific features of a single-pulse sliding discharge in neon near the threshold for spark breakdown

Experimental data on the spatial structure of a single-pulse sliding discharge in neon at voltages below, equal to, and above the threshold for spark breakdown are discussed. The experiments were carried at gas pressures of 30 and 100 kPa and different polarities of the discharge voltage. Photographs of the plasma structure in two discharge chambers with different dimensions of the discharge zone and different thicknesses of an alumina dielectric plate on the surface of which the discharge develops are inspected. Common features of the prebreakdown discharge and its specific features depending on the voltage polarity and gas pressure are analyzed. It is shown that, at voltages below the threshold for spark breakdown, a low-current glow discharge with cathode and anode spots develops in the electrode gap. Above the breakdown threshold, regardless of the voltage polarity, spark channels directed from the cathode to the anode develop against the background of a low-current discharge.     

On the relation between the index of the interelectrode gap filling with spark channels and the current of a pulsed multichannel sliding discharge in Ne,Ar, and Xe

Experimental results and model concepts concerning the relation between the index K of the interelectrode gap filling with spark channels and the peak current I _peak of a single-pulse submicrosecond multichannel complete sliding discharge on an alumina ceramic surface are discussed. The spatial structure of an incomplete discharge at the threshold for the surface spark breakdown of gas is considered. The experiments were performed with three gases, Ne, Ar, and Xe, at pressures of 30 and 100 kPa and opposite polarities of the discharge voltage, with two discharge chambers differing in the geometry of the discharge gap and the thickness of the ceramic plate. It is shown that, although the structure of the incomplete discharge at the threshold for spark breakdown varies from diffuse homogeneous to pronounced filamentary, the dependence \(K\left( {\sqrt[6]{{I_{peak} }}} \right)\) for a complete discharge is close to linear and can be qualitatively explained by the earlier proposed semiempirical model of the time evolution of the structure of a multichannel discharge. In particular, the estimated steepness of the dependence \(K\left( {\sqrt[6]{{I_{peak} }}} \right)\) agrees best with the experimental results when the local density of free electrons at the threshold for spark breakdown is 10¹⁶ cm^?3 or higher.     

New faces in plant innate immunity: heterotrimeric G proteins

Co-existence of species seems to inevitably result in origin of parasitism and hence development of molecular mechanisms of attack and defense. Certain similarities between plant and animal defense systems point to an ancient inheritance of the innate immunity. Heterotrimeric G proteins are structurally conserved signaling molecules connecting plasma membrane bound receptors to cytoplasmic effectors. They were found in most eukaryotic organisms. Their role in human pathophysiology and animal diseases was well established. In plants these proteins were also recently implicated in innate immunity. However, molecular mechanisms governed by G proteins and providing resistance against plant pathogens seem to be different from those in animal systems and largely remain elusive. In this review we attempted to sketch current ideas of plant defense system and to present a contemporary status of heterotrimeric G proteins in plant innate immunity.     

Dissecting Arabidopsis Gβ signal transduction on the protein surface

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits.     

Silencing of the ACC synthase gene ACACS2 causes delayed flowering in pineapple [Ananas comosus (L.) Merr.

Flowering is a crucial developmental stage in the plant life cycle. A number of different factors, from environmental to chemical, can trigger flowering. In pineapple, and other bromeliads, it has been proposed that flowering is triggered by a small burst of ethylene production in the meristem in response to environmental cues. A 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) gene has been cloned from pineapple (ACACS2), which is induced in the meristem under the same environmental conditions that induce flowering. Two transgenic pineapple lines have been produced containing co-suppression constructs designed to down-regulate the expression of the ACACS2 gene. Northern analysis revealed that the ACACS2 gene was silenced in a number of transgenic plants in both lines. Southern hybridization revealed clear differences in the methylation status of silenced versus non-silenced plants by the inability of a methylation-sensitive enzyme to digest within the ACACS2 DNA extracted from silenced plants, indicating that methylation is the cause of the observed co-suppression of the ACACS2 gene. Flowering characteristics of the transgenic plants were studied under field conditions in South East Queensland, Australia. Flowering dynamics studies revealed significant differences in flowering behaviour, with transgenic plants exhibiting silencing showing a marked delay in flowering when compared with non-silenced transgenic plants and control non-transformed plants. It is argued that the ACACS2 gene is one of the key contributors towards triggering 'natural flowering' in mature pineapples under commercial field conditions.     

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gβ, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant‐specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C–terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub‐population of Gβ/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine‐rich extracellular C–terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine‐rich C–terminus is extracellular.