XCC

Disturbance of Arabidopsis thaliana microRNA-regulated pathways by Xcc bacterial effector proteins.[Pubmed:24385242]

Amino Acids. 2014 Apr;46(4):953-61.

Plants are continuously subjected to infection by pathogens, including bacteria and viruses. Bacteria can inject a variety of effector proteins into the host to reprogram host defense mechanism. It is known that microRNAs participate in plant disease resistance to bacterial pathogens and previous studies have suggested that some bacterial effectors have evolved to disturb the host's microRNA-regulated pathways; and so enabling infection. In this study, the inter-species interaction between an Xanthomonas campestris pv campestris (XCC) pathogen effector and Arabidopsis thaliana microRNA transcription promoter was investigated using three methods: (1) interolog, (2) alignment based on using transcription factor binding site profile matrix, and (3) the web-based binding site prediction tool, PATSER. Furthermore, we integrated another two data sets from our previous study into the present web-based system. These are (1) microRNA target genes and their downstream effects mediated by protein-protein interaction (PPI), and (2) the XCC-Arabidopsis PPI information. This present work is probably the first comprehensive study of constructing pathways that comprises effector, microRNA, target genes and PPI for the study of pathogen-host interactions. It is expected that this study may help to elucidate the role of pathogen-host interplay in a plant's immune system. The database is freely accessible at: http://ppi.bioinfo.asia.edu.tw/EDMRP .

Xcc-facilitated agroinfiltration of citrus leaves: a tool for rapid functional analysis of transgenes in citrus leaves.[Pubmed:25146436]

Plant Cell Rep. 2014 Dec;33(12):1993-2001.

KEY MESSAGE: Xanthomonas citri subsp. citri pretreatment before agroinfiltration could significantly promote transient expression in citrus leaves which were previously recalcitrant to agroinfiltration. Transient expression via agroinfiltration is widely used in biotechnology but remains problematic in many economically important plants. Xanthomonas citri subsp. citri (XCC)-facilitated agroinfiltration was employed to promote transient protein expression in Valencia sweet orange leaves, which are recalcitrant to agroinfiltration. However, it is unclear whether XCC-facilitated agroinfiltration has broad application, i.e., whether XCC-facilitated agroinfiltration could be used on other citrus varieties. In addition, we intended to investigate whether XCC-facilitated agroinfiltration could be used to hasten transgene function assays, e.g., Cre/lox system and Cas9/sgRNA system. In this report, XCC-facilitated agroinfiltration was further exploited to enhance beta-glucuronidase (GUS) expression in five citrus varieties. XCC-facilitated agroinfiltration also significantly increased GFP expression in six citrus varieties tested. Both GUS and GFP assays indicated that XCC-facilitated agroinfiltration had the best performance in grapefruit. After XCC-facilitated agroinfiltration was carried out in grapefruit, protoplast analysis of the transformed cells indicated that there were more than 20 % leaf cells expressing GFP. In grapefruit, usefulness of XCC-facilitated agroinfiltration was assayed in three case studies: (1) fast functional analysis of Cre/lox system, (2) the heat shock regulation of HSP70B promoter derived from Arabidopsis, and (3) Cas9/sgRNA-mediated genome modification.

Autodisplay of an archaeal gamma-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.[Pubmed:24756321]

Appl Microbiol Biotechnol. 2014 Aug;98(16):6991-7001.

At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter XCC_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of XCC_Est as an anchoring motif for surface display of gamma-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of gamma-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated XCC_Est could efficiently transport gamma-lactamase to the cell surface. Compared with the free enzyme, the displayed gamma-lactamase exhibited optimum temperature of 30 degrees C other than 90 degrees C, with a substantial decrease of 60 degrees C. Under the preparation system, the engineered E. coli with autodisplayed gamma-lactamase converted 100 g racemic vince lactam to produce 49.2 g (-) vince lactam at 30 degrees C within 4 h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed gamma-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.

Lactose consuming strains of Xanthomonas citri subsp. citri (Xcc) insight into the emergence of natural field resources for xanthan gum production.[Pubmed:24318518]

World J Microbiol Biotechnol. 2014 May;30(5):1511-7.

Xanthomonas genus possesses a low level of beta-galactosidase gene expression and is therefore unable to produce xanthan gum in lactose-based media. In this study, we report the emergence of some natural field strains of Xanthomonas citri subsp. citri (XCC) capable to use lactose as a sole carbon source to produce xanthan gum. From 210 XCC strains isolated from key lime (C. aurantifolia), 27 showed the capacity to grow on lactose containing medium. XCC lactose consuming strains demonstrated a good level of xanthan production. Amongst all, NIGEBK37 produced the greatest (14.62 g/l) amount of xanthan gum in experimental laboratory conditions. By evaluating the viscosity of the biopolymer at 25 degrees C, it was demonstrated that xanthan synthesized by strain NIGEBK37 has the highest viscosity (44,170.66 cP). Our results were indicative for the weakness of a commercial strain of Xanthomonas campestris pv. Campestris DSM1706 (XCC/DSM1706) to produce xanthan in lactose containing medium.

Adenosine receptor antagonists induce persistent bursting in the rat hippocampal CA3 region via an NMDA receptor-dependent mechanism.[Pubmed:10758091]

J Neurophysiol. 2000 Apr;83(4):1787-95.

Adenosine receptor antagonists initiate repetitive bursting activity in the CA3 region of hippocampal slices. Although some studies have suggested that this effect is irreversible, this has been difficult to establish because many adenosine antagonists wash out of brain slices extremely slowly. Furthermore the cellular mechanism that underlies persistent bursting is unknown. To resolve these issues, we studied the effects of nonselective (8-p-sulfophenyltheophylline, 8SPT, 50-100 microM), A(l)-selective (8-cyclopentyl-1, 3-dipropylxanthine, 100 nM; xanthine carboxylic acid congener, 200 nM), and A(2A)-selective (chlorostyryl-caffeine; 200 nM) adenosine antagonists in the CA3 region of rat hippocampal slices using extracellular recording. Superfusion with all of the adenosine antagonists except chlorostyryl-caffeine induced bursting, and the burst frequency after 30 min drug superfusion did not differ for the different antagonists. Most slices showed a period of rapid initial bursting, followed either by stable bursting at a lower frequency or a pattern of oscillating burst frequency. In either case, the bursting continued after drug washout. Virtually identical patterns of long-term bursting activity were observed when 8SPT was washed out or applied continuously. Control experiments using exogenous adenosine to characterize the persistence of 8SPT in tissue demonstrated >95% washout at 60 min, a time when nearly all slices still showed regular bursting activity. When the N-methyl-D-aspartate (NMDA) antagonists DL-2-amino-5-phosphonovaleric acid (AP5; 50 microM) or dizocilpine (10 microM) were applied before and during 8SPT superfusion, bursting occurred in the presence of the NMDA antagonists but did not persist once the 8SPT was washed out. AP5 had no effect on persistent bursting when applied after the initiation of spiking. The selective calcium/calmodulin-dependent protein kinase inhibitor 1-[N, O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62; 3 microM), which has been shown to block NMDA receptor-dependent synaptic plasticity in the CA1 region, also significantly decreased the long-term effect of 8SPT. Thus adenosine antagonists initiate persistent spiking in the CA3 region; this activity does not depend on continued occupation of adenosine receptors by antagonists, and can be blocked by treatments that prevent NMDA receptor-dependent plasticity.

The inhibitory adenosine receptor at the neuromuscular junction and hippocampus of the rat: antagonism by 1,3,8-substituted xanthines.[Pubmed:2257444]

Br J Pharmacol. 1990 Oct;101(2):453-9.

1. The ability of 1,3,8-substituted xanthines to antagonize the inhibitory effects of adenosine receptor agonist on the amplitude of nerve-evoked twitches of the rat phrenic-diaphragm and on the amplitude of orthodromically-evoked population spikes, recorded from the CA1 pyramidal cells of rat hippocampal slices, was investigated. 2. 1,3-Dipropyl-8-cyclopenthylxanthine (DPCPX), 1,3-dipropyl-8-(carboxymethyloxyphenyl)xanthine (XCC), 1,3-dipropyl-8-(4-[2-aminoethyl)amino)carbonylmethyloxyphenyl)x ant hine (XAC), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), 8-phenyltheophylline (8-PT), 1,3-diethyl-8-phenylxanthine (DPX) and PD 115,199, in concentrations virtually devoid of effect on neuromuscular transmission, shifted to the right in a near parallel manner the log concentration-response curve for the inhibitory effect of 2-chloroadenosine (CADO) on nerve-evoked twitches of the phrenic-diaphragm. Linear Schild plots with slopes near to unity were obtained for all the xanthines. 3. The order of potency of the xanthines as antagonists of the effect of CADO in the phrenic-diaphragm was DPCPX (Ki = 0.54 nM) greater than XCC (Ki = 10 nM), XAC (Ki = 11 nM), PACPX (Ki = 13 nM) greater than DPX (Ki = 22 nM), 8-PT (Ki = 25 nM) greater than PD 115,199 (Ki = 57 nM). The potency of DPCPX in antagonizing the inhibitory effects of R-N6-phenylisopropyladenosine (R-PIA) and 5'-N-ethylcarboxamide adenosine (NECA) on nerve-evoked twitch response was not statistically different from its potency in antagonizing the inhibitory effect on CADO. 4. In the hippocampal slices, DPCPX, XCC and XAC, used in concentrations virtually devoid of effect on population spike amplitude, shifted to the right in a near parallel manner the log concentrationresponse curve for the inhibitory effect of CADO on the amplitude of the population spikes. The Schild plots were linear with slopes near unity. 5. The potencies of DPCPX (K, = 0.45 nM) and XAC (K, = 11 nM) in antagonizing the inhibitory adenosine receptor at the hippocampus were similar to their potencies for antagonism of the inhibitory adenosine receptor at the phrenic-diaphragm. XCC was only slightly more potent (K, = 5.4 nM) as an antagonist of the adenosine receptor in the hippocampus than in the phrenic-diaphragm. 6. The results suggest that the inhibitory adenosine receptors in the phrenic-diaphragm and in the hippocampus of the rat are similar, and that according to the antagonist potencies, these receptors belong to the A1-adenosine receptor subtype.

Autoradiographic characterization of high-affinity adenosine A2 receptors in the rat brain.[Pubmed:2713675]

Brain Res. 1989 Apr 10;484(1-2):111-8.

Binding of the non-selective adenosine receptor agonist, [3H]NECA (5'-N-ethylcarboxamidoadenosine) was evaluated in sections of rat brain using quantitative receptor autoradiography. [3H]NECA bound specifically to a variety of different brain regions including striatum, cerebellum and thalamus. In the presence of the selective adenosine A1 receptor agonist, cyclopentyladenosine (CPA: 50 nM), [3H]NECA binding was exclusively localized to the striatum and olfactory tubercle. Binding in rat striatum occurred at a single site (Kd = 9 nM) with limited capacity (apparent Bmax = 230 fmol/mg tissue). Competition experiments in both striatum and olfactory tubercle with various adenosine agonists and antagonists indicated that the sites labeled by [3H]NECA in the presence of 50 nM CPA were A2 in nature, the rank order of activity for agonists being NECA greater than 2-chloroadenosine (2-CADO), greater than R-N6-phenylisopropyladenosine (R-PIA) greater than CPA greater than S-N6-phenylisopropyladenosine (S-PIA). For xanthine antagonists the order was greater than 1,3-dipropyl-8(2-amino-4-chloro)phenylxanthine (PACPX) greater than xanthine amino acid congener (XAC) greater than xanthine carboxylic acid congener (XCC) greater than 1,3-diethyl-8-phenylxanthine (DPX). The localization of A2 receptors to discrete regions of rat brain indicates that the purine may have a selective role in modulating basal ganglia function.