Do Plants Contain G Protein-Coupled Receptors?

Whether G protein-coupled receptors (GPCRs) exist in plants is a fundamental biological question. Interest in deorphanizing new GPCRs arises because of their importance in signaling. Within plants, this is controversial, as genome analysis has identified 56 putative GPCRs, including G protein-coupled receptor1 (GCR1), which is reportedly a remote homolog to class A, B, and E GPCRs. Of these, GCR2 is not a GPCR; more recently, it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix-alignment method, which has been benchmarked against the class A-class B-class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologs to class A, class B, and class F GPCRs and shown that GCR1 is closer to class A and/or class B GPCRs than class A, class B, or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the six GPCR classes. Variability comparisons provide additional evidence that GCR1 homologs have the GPCR fold. From the alignments and a GCR1 comparative model, we have identified motifs that are common to GCR1, class A, B, and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fold.There has been much interest in the identification of novel G protein-coupled receptors (GPCRs) from genome analysis, initially from the human genome, because GPCRs are highly druggable therapeutic targets, and more recently from other genome studies, because GPCRs are vital signaling molecules in diverse organisms. Therefore, whether GPCRs exist in plants is a fundamental biological question.Here, our focus on putative plant GPCRs was initiated with the characterization of G protein-coupled receptor1 (GCR1) as an orphan GPCR that binds to the plant G protein Guanine nucleotide-binding protein alpha-1 subunit (GPA1) and that is involved in the drought response (Hooley, 1999; Pandey and Assmann, 2004). This observation was followed by intense efforts to identify other plant GPCRs (Moriyama et al., 2006; Liu et al., 2007; Gookin et al., 2008; Pandey et al., 2009). For well-established GPCRs, there are two main classification systems. The GRAFS system (Fredriksson et al., 2003) described five classes of human GPCRs: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin. Others (Attwood and Findlay, 1994; Kolakowski, 1994) described six classes, namely A to E and the Frizzled GPCRs (class F), that additionally include class D (Eilers et al., 2005) found in fungi and class E cAMP receptors associated with Dictyostelium species (Williams et al., 2005); the Adhesion and Secretin receptors, which differ primarily in their N termini (Lagerström and Schiöth, 2008), together form class B. GCR1 is particularly interesting from a bioinformatics perspective, as it has identifiable but distant homology to class E, class B, and class A GPCRs (Pandey and Assmann, 2004), and so has been used to inform the medically important class A-class B GPCR alignment (Vohra et al., 2007, 2013). GCR1 and the other putative plant GPCRs do not naturally fall into the well-characterized GPCR classes, as presented at the GPCRDB (Horn et al., 2003; Vroling et al., 2011) or elsewhere, and so confirmation that GCR1 is a GPCR is difficult. Indeed, the pitfalls of GPCR identification are illustrated by the high profile (Liu et al., 2007) but erroneous identification of GCR2 as a plant GPCR. It has now been confirmed through crystallization that GCR2 is a lantibiotic cyclase-like protein (Chen et al., 2013), as predicted by our fold recognition studies (Illingworth et al., 2008).We are particularly interested in these putative GPCRs to assess whether, as remote homologs, they may similarly be used to address the difficult issue of alignment between GPCR families. In this respect, only GCR1 is useful, as the fold recognition studies indicate that GCR1 is the most likely candidate to have a GPCR fold while the evidence for other plant GPCRs is at best minimal. While many methods have been used to align GPCRs from different classes (Frimurer and Bywater 1999; Sheikh et al., 1999; Bissantz et al., 2004; Miedlich et al., 2004; Eilers et al., 2005; Kratochwil et al., 2005; Dong et al., 2007; Coopman et al., 2011; Gregory et al., 2013), it has not been possible to validate these methods on GPCRs until recently. However, with the recent publication of the structure of the class B glucagon receptor (Siu et al., 2013), the class B corticotropin-releasing factor1 receptor (Hollenstein et al., 2013), and the class F human smoothened receptor (Wang et al., 2013) and the associated structural alignments between class A and these remote homologs, we have been able, to our knowledge for the first time, to successfully test our new method. This method is a variation on that used to produce a well-validated class A-class B alignment (Vohra et al., 2013), in which GCR1 was used as a bridge; in a follow-on article, the alignment formed the basis of a class B calcitonin receptor-like receptor (CLR) active model (Woolley et al., 2013) that was later shown to be in good agreement with the class B glucagon receptor x-ray crystal structure. Consequently, we have aligned the GCR1 homologs to class A, class B, and class F and have generated comparative models of active and inactive GCR1. From the alignment, with the assistance of the models, we have identified a number of motifs that are common to GCR1, class A, class B, and class E GPCRs, thus greatly increasing the evidence that GCR1 has a GPCR fold. In addition, we have provided further evidence that GCR1 homologs have the same fold as class A and class B GPCRs from variability analysis. Here, we imply that the difference between a GPCR and a protein with a GPCR fold is the lack of definitive experimental evidence of conventional signaling partners.Some bioinformatics studies have suggested that there might be about 50 plant GPCRs, including those with large non-membrane domains (Supplemental Table S1) or those with sequence similarities to other plant proteins (Supplemental Table S2), but now it has been questioned whether there are any plant GPCRs (Urano et al., 2012, 2013; Bradford et al., 2013; Urano and Jones 2013), primarily because the plant G protein is self activating and does not need a guanine nucleotide-exchange factor (GEF). One of the presentations of putative plant GPCRs is based on a hidden Markov model, trained on several hundred seven-transmembrane helical (7TM) proteins taken from the GPCRDB (both well-characterized GPCRs and other 7TM proteins such as the mildew resistance locus O [MLO] proteins); the genes were tentatively assigned as GPCRs on the basis of seven predicted transmembrane helices (Moriyama et al., 2006). This assignment has been made against the background of the well-documented and now closed debate regarding whether the 7TM protein bacteriorhodopsin was a suitable template for modeling GPCRs (Hibert et al., 1993; Hoflack et al., 1994), most typified by the article of Hibert et al. (1993): “This is not a G protein coupled receptor.” Given that a number of distinct GPCR x-ray crystal structures have become available (Congreve et al., 2011; Katritch et al., 2013; Venkatakrishnan et al., 2013), it is now possible to analyze these putative plant GPCR sequences to assess whether, in the light of new structural information, they are more or less likely to be GPCRs and, thus, to move beyond the assumption implicit in Moriyama et al. (2006) that a receptor with seven transmembrane helices is a GPCR (e.g. bacteriorhodopsin has seven transmembrane helices but is not a GPCR; Hibert et al., 1993).Here, our approach to analysis of the 56 putative plant GPCRs is to combine transmembrane structure prediction and sequence analysis with fold recognition methods. There are essentially two approaches to fold recognition, namely sequence-based methods, such as genTHREADER (Jones, 1999b), and empirical potential-based methods, such as Threader (Jones et al., 1992; Jones, 1998). The sequence-based methods have the advantage of speed and may be suitable for whole-genome analysis but may not readily identify remote homologs when the sequence identity is low. The empirical potential-based methods may be more efficient at identifying remote homologs but are generally not parameterized for membrane proteins. For this reason, we have taken a heuristic approach and have tested a variety of fold recognition methods to see if they correctly identify characteristic GPCR sequences from classes A to F while at the same time not incorrectly assigning bacteriorhodopsin and GCR2 as GPCRs. In particular, our focus is on fold recognition methods such as I-TASSER (Zhang, 2008; Roy et al., 2010) that have performed well in the critical assessment of protein structure prediction (CASP) fold recognition competitions (Moult et al., 2009). For proteins where the evidence that they are GPCRs was not convincing, the fold recognition (threading) results were used to give a preliminary indication of which other types of membrane proteins they could be; the most likely alternatives were ion channels or transporters. The significance of this study, therefore, is 4-fold. First, it adds clarity to the field of plant GPCRs by indicating from a wide range of evidence that only GCR1 is strongly predicted to have a GPCR fold. Second, it provides evidence that some of the other candidates are more likely to be transporters. Third, it indicates computational approaches that could be taken to follow up initial genome analysis studies to help avoid the confusion that has shrouded the plant GPCR field. Fourth, the new alignment method has given promising results on well-validated alignments in or below the “twilight zone” (Doolittle, 1986) and so could, with development, be used in other more general applications. In addition, we discuss the implications of these results that are difficult to reconcile with current knowledge of the mechanism of the Arabidopsis (Arabidopsis thaliana) G protein, GPA1.     

Gastroprotective Activity of Ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylidene) Amino]benzoate against Ethanol-Induced Gastric Mucosal Ulcer in Rats

Background

The study was carried out to determine the cytotoxic, antioxidant and gastro-protective effect of ethyl-4-[(3,5-di-tert-butyl-2-hydroxybenzylid ene)amino] benzoate (ETHAB) in rats.

Methodology/Principal Findings

The cytotoxic effect of ETHAB was assessed using a MTT cleavage assay on a WRL68 cell line, while its antioxidant activity was evaluated in vitro. In the anti-ulcer study, rats were divided into six groups. Group 1 and group 2 received 10% Tween 20 (vehicle). Group 3 received 20 mg/kg Omeprazole. Groups 4, 5 and 6 received ETHAB at doses of 5, 10, and 20 mg/kg, respectively. After an hour, group 1 received the vehicle. Groups 2–6 received absolute ethanol to induce gastric mucosal lesions. In the WRL68 cell line, an IC₅₀ of more than 100 µg/mL was observed. ETHAB results showed antioxidant activity in the DPPH, FRAP, nitric oxide and metal chelating assays. There was no acute toxicity even at the highest dosage (1000 mg/kg). Microscopy showed that rats pretreated with ETHAB revealed protection of gastric mucosa as ascertained by significant increases in superoxide dismutase (SOD), pH level, mucus secretion, reduced gastric lesions, malondialdehyde (MDA) level and remarkable flattened gastric mucosa. Histologically, pretreatment with ETHAB resulted in comparatively better gastric protection, due to reduction of submucosal edema with leucocyte infiltration. PAS staining showed increased intensity in uptake of Alcian blue. In terms of immunohistochemistry, ETHAB showed down-expression of Bax proteins and over-expression of Hsp70 proteins.

Conclusion/Significance

The gastroprotective effect of ETHAB may be attributed to antioxidant activity, increased gastric wall mucus, pH level of gastric contents, SOD activity, decrease in MDA level, ulcer area, flattening of gastric mucosa, reduction of edema and leucocyte infiltration of the submucosal layer, increased PAS staining, up-regulation of Hsp70 protein and suppressed expression of Bax. Key words: ethyl 4-(3, 5-di-ter-butyl-2-hydroxybenzylamino) benzoate; toxicity; antioxidant; gastric-ulcer; anti-ulcer; histology; immunohistochemistry.     

Evaluation of Chemopreventive Effects of Acanthus ilicifolius against Azoxymethane-Induced Aberrant Crypt Foci in the Rat Colon

Background

Acanthus ilicifolius, a mangrove medicinal plant, is traditionally used to treat a variety of diseases. The aim of this research is to assess the chemoprotective outcomes of A. ilicifolius ethanolic extract against azoxymethane (AOM) induced colonic aberrant crypt foci (ACF) in rats.

Methodology/Principal Findings

In our study, rats were arranged in to five groups. Rats in the normal control group were given subcutaneous injections of normal saline once weekly for 2 weeks. The AOM control, reference and treatment groups were given subcutaneous injection of AOM, 15 mg/kg body weight, once weekly for 2 weeks each. The reference group was treated with 35 mg/kg 5-Fluorouracil via intraperitoneal injection once weekly for 8 weeks, and the treatment groups were administered by gavage with 250 and 500 mg/kg A. ilicifolius extract daily for 8 weeks. Both normal and AOM control groups received the vehicle; 10% Tween-20 only.Rats treated with 250 mg/kg and 500 mg/kg of A. ilicifolius extracts showed a decrease in the mean number of ACF by 65% and 53%, respectively. Those fed with A. ilicifolius showed significantly decreased multiplicity of ACF formations when compared with the results from the AOM control group. The 250 mg/kg A. ilicifolius treatment group showed significant decreases in lipid peroxidation MDA levels when compared with the AOM control group. In immunohistochemistry staining, the proliferating nuclear cell antigen (PCNA)-positive cells were significantly higher in the AOM control group than in the A. ilicifolius-treated groups. RT-PCR showed that A. ilicifolius caused a change in the regulation of apoptosis-related genes expression.

Conclusion/Significance

The results of the current study show that AOM-treated rats receiving oral exposure to A. ilicifolius demonstrated a significant decrease in the number of ACF in the colon when compared to AOM-treated rats receiving vehicle only. A ilicifolius may be an effective herbal approach for the prevention of AOM-induced ACF in the rat colon.     

Chemopreventive Efficacy of Andrographis paniculata on Azoxymethane-Induced Aberrant Colon Crypt Foci In Vivo

Andrographis paniculata is a grass-shaped medicinal herb, traditionally used in Southeast Asia. The aim of this study was to evaluate the chemoprotective effects of A. paniculata on colorectal cancer. A. paniculata ethanol extract was tested on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in vivo and in vitro. A. paniculata treated groups showed a significant reduction in the number of ACF of the treated rats. Microscopically, ACF showed remarkably elongated and stratified cells, and depletion of the submucosal glands of AOM group compared to the treated groups. Histologically, staining showed slightly elevated masses above the surrounding mucosa with oval or slit-like orifices. Immunohistochemically, expression of proliferating cell nuclear antigen (PCNA) and β-catenin protein were down-regulated in the A. paniculata treated groups compared to the AOM group. When colon tissue was homogenized, malondialdehyde (MDA) and nitric oxide (NO) levels were significantly decreased, whereas superoxide dismutase (SOD) activity was increased in the treated groups compared to the AOM group. A. paniculata ethanol extract showed antioxidant and free radical scavenging activity, as elucidated by the measure of oxidative stress markers. Further, the active fractions were assessed against cell lines of CCD841 and HT29 colon cancer cells.     

Role of Serine Proteases in the Regulation of Interleukin-877 during the Development of Bronchopulmonary Dysplasia in Preterm Ventilated Infants

Rationale

The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-8₇₇) is a less potent chemoattractant than other shorter isoforms. Although interleukin-8₇₇ is abundant in the preterm circulation, its regulation in the preterm lung is unknown.

Objectives

To study expression and processing of pulmonary interleukin-8₇₇ in preterm infants who did and did not develop bronchopulmonary dysplasia.

Methods

Total interleukin-8 and interleukin-8₇₇ were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function.

Main Results

Peak total interleukin-8 and interleukin-8₇₇ concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-8₇₇ to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-8₇₇ to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01).

Conclusions

Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-8₇₇ by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.     

新疆小孢发属地衣的分类

根据多年的野外调查和相关的研究资料,对小孢发属(BryoriaB rodo & Hawksw.)地衣进行了分类学研究,报道新疆小孢发属地衣10种。其中Bryoria chalybeiformis,Bryoria fuscescens,Bryoria nadvornikiana和Bryo-riasimplicior为中国新记录种,Bryoria lanestris,Bryoria nitidula,Bryoria trichodes ssp.trichodes,Bryoria pseudo-fuscescens为新疆新记录种。     

Inhibition of suicidal erythrocyte death by pyrogallol

Molecular Biology Reports - Pyrogallol, a polyphenolic component of Acacia nilotica has previously been reported to induce apoptosis of diverse cell types. Pyrogallol is in part effective by...     

Effect of boundary type and season on predatory arthropods associated with field margins on New Zealand farmland

Pitfall traps were used to monitor predatory arthropod numbers along two types of field boundary, a post and wire fence line and a Cupressus macrocarpa hedge, along the same paddock margin in Canterbury, New Zealand, over 24 months. The seven most abundant predator groups recorded were: Araneae > Phalangiidae > Staphylinidae > Coccinellidae > Chilopoda > Hemerobiidae > Carabidae. Araneae, Phalangiidae, Staphylinidae, Chilopoda and Hemerobiidae were found in larger numbers at the wire fence than at the hedge site, whereas the numbers of Carabidae and Coccinellidae adults exhibited no field margin preference. However, more species of Araneae and Staphylinidae were caught at the hedge site, whereas species richness of carabid beetles was greatest at the wire fence. Principal component analysis clearly separated the samples collected from the two habitats based on the assemblages of Araneae, Staphylinidae and Carabidae, and certain species in each of these taxonomic groups appeared to be particularly associated with one boundary type or the other. All the main taxonomic groups exhibited clear seasonal patterns, with distinct peaks in abundance occurring at certain times of the year. The results of the study reinforce the idea that management of field boundaries can be used to manipulate the type and abundance of particular groups of predatory arthropods, and that seasonal patterns should be taken into account in schemes of integrated pest management so that any adverse effects of biocide application on these beneficial species may be minimised.     

Enhancement of rat brain catecholamine synthesis by administration of small doses of tyrosine and evidence for substrate inhibition of tyrosine hydroxylase activity by large doses of the amino acid

Rat brain catecholamine synthesis is enhanced by small doses of tyrosine, but not by doses of 50mg/kg body wt. and above. It is suggested that these latter doses overcome the above enhancement by causing a substrate inhibition of tyrosine hydroxylase activity.