bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of swarming and virulence factor expression in Proteus mirabilis by resveratrol
Authors: Wang Won-Bo , Lai Hsin-Chih , Hsueh Po-Ren , Chiou Robin Y.-Y. , Lin Shwu-Bin , Liaw Shwu-Jen
Published: September 2006

Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a phytoalexin compound with anti-inflammatory and antioxidant activities. The effect of resveratrol on swarming and virulence factor expression of Proteus mirabilis, an important pathogen infecting the urinary tract, was determined on swarming agar plates with and without the compound. Bacteria harvested at different times were assayed for cell length and the production of flagella, haemolysin and urease. Resveratrol inhibited P. mirabilis swarming and virulence factor expression in a dose-dependent manner. Resveratrol significantly inhibited swarming at 15 μg ml−1, and completely inhibited swarming at 60 μg ml−1. Inhibition of swarming and virulence factor expression was mediated through RsbA, a His-containing phosphotransmitter of the bacterial two-component signalling system possibly involved in quorum sensing. Complementation of an rsbA-defective mutant with the rsbA gene restored its responsiveness to resveratrol. The compound also inhibited the ability of P. mirabilis to invade human urothelial cells. These findings suggest that resveratrol has potential to be developed as an antimicrobial agent against P. mirabilis infection.
Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis
Authors: Liu Binbo , Liu Shengwu , Qu Xueju , Liu Junyan
Published: September 2006

A full-length cDNA of granulysin was inserted into the pcDNA3.1(−) vector to construct a eukaryotic expression plasmid for granulysin. The recombinant plasmids were injected intramuscularly into mice infected withMycobacterium tuberculosisto evaluate the protective effect of granulysin. Granulysin significantly decreased the weight index (WI) of the spleen, reduced the numbers of viable bacteria in lung and spleen, and reduced the lesions of lung tissue in granulysin-rDNA-immunized mice compared with those of control group mice.In vitro, the serum of the recombinant-plasmid-immunized mice inhibited the viability ofM. tuberculosisby the physical disruption of cell membranes. Therefore, granulysin has a therapeutic effect againstM. tuberculosis.
Frequency and predictors of colonization of the respiratory tract by VIM-2-producing Pseudomonas aeruginosa in patients of a newly established intensive care unit
Authors: Horianopoulou Maria , Legakis Nicholas J. , Kanellopoulou Maria , Lambropoulos Sotiris , Tsakris Athanassios , Falagas Matthew E.
Published: September 2006

The aim of this study was to examine the frequency and predictors of colonization of the respiratory tract by metallo-β-lactamase (MBL)-producing Gram-negative bacteria in patients admitted to a newly established intensive care unit (ICU) of a tertiary care hospital. Specimens of tracheobronchial aspirates for microbiological studies were obtained every day for the first 3 days of the ICU stay and subsequently every third day for the rest of the ICU stay. PCR analysis and nucleotide sequencing were performed to identify bacteria that had MBL genes. Thirty-five patients (20 male, 15 female) were hospitalized during the initial 3 month period of functioning of the ICU. Colonization of the lower respiratory tract by Gram-negative bacteria was found in 29 of 35 patients (83 %) during the first 6–20 days (median 13 days) following admission to the ICU (13 patients with Acinetobacter baumannii, ten with Pseudomonas aeruginosa, three with Enterobacter aerogenes, two with Klebsiella pneumoniae and one with Stenotrophomonas maltophilia). Six of 29 patients (21 %) colonized with Gram-negative bacteria had bla
VIM-2-positive P. aeruginosa isolates; one of these patients developed clinical infection due to this micro-organism. Previous use of carbapenems (P=0.01) or other β-lactams (P=0.03), as well as a stay in the ICU of >20 days (P<0.001), were associated with colonization with bla
VIM-2-producing P. aeruginosa. In conclusion, colonization by Gram-negative bacteria of the respiratory tract of patients in this newly established ICU was common (83 %). Use of β-lactams, including carbapenems, was associated with subsequent colonization of the respiratory tract with MBL-positive P. aeruginosa.
Evaluation of a glycerol-preserved antigen in the direct agglutination test for diagnosis of visceral leishmaniasis at rural level in eastern Sudan
Authors: el Mutasim Mohamed , Mansour Durria , Abass Elfadil M. , Hassan Wisam M. , el Harith Abdallah
Published: September 2006

Three-hundred and eight patients with suspected visceral leishmaniasis (VL) were received at Doka Hospital (eastern Sudan) during the period September 2004 to October 2005. The sensitivity and specificity of a glycerol-preserved (GP) antigen for VL diagnosis was assessed against the results of repeated lymph node aspiration and readings from a direct agglutination test (DAT) employing standard formaldehyde-fixed (FF) or freeze-dried (FD) antigen. Despite 13 months of storage at ambient temperature (28–47 °C), the GP antigen mean titres obtained from these 308 patients were no different from those that were FD (P=0.945) and stored under similar conditions, but were significantly different (P=0.019) from those that were FF and kept continuously at the optimum temperature for storage (4–8 °C). Taking the parasitological result as the gold standard and using a pre-established titre of 1 : 3200 as the DAT cut-off, the GP antigen revealed a sensitivity (91/105, 86.7 %) and specificity (187/203, 92.1 %) comparable to that of FD antigen (92/105, 87.6 %, and 188/203, 92.6 %, respectively) and FF antigen (94/105, 89.5 %, and 188/203, 92.6 %, respectively). At a titre range of 1 : 400–1 : 800, statistically determined as the optimum cut-off for the three antigens, sensitivities of 92.4, 90.5 and 96.2 % and specificities of 90.6, 90.1 and 88.7 % were achieved for the GP, FD and FF antigens, respectively, at a peripheral hospital. Regardless of the antigen preparation used, DAT results obtained in the peripheral hospital were highly reproducible in the central laboratory in Omdurman (weighted kappa: GP=0.957, FD=0.979 and FF=0.936). With a diagnostic reliability comparable to formaldehyde fixation and stability under ambient conditions similar to freeze drying, glycerol preservation, by virtue of its high potential for reproduction, meets the requirements for the management of VL in developing countries.
RepAM of the Amycolatopsis methanolica integrative element pMEA300 belongs to a novel class of replication initiator proteins
Authors: te Poele Evelien M. , Kloosterman Harm , Hessels Gerda I. , Bolhuis Henk , Dijkhuizen Lubbert
Published: September 2006

Accessory genetic elements, such as plasmids and integrative elements, are widespread amongst actinomycetes, but little is known about their functions and mode of replication. The conjugative element pMEA300 from Amycolatopsis methanolica is present mostly in an integrated state at a single specific site in the chromosome, but it can also replicate autonomously. Complete nucleotide sequencing, in combination with deletion studies, has revealed that orfB of pMEA300 is essential for autonomous replication in its host. In this study, it was shown that purified OrfB protein binds specifically to the 3′ end of its own coding sequence. Within this short sequence, a putative hairpin structure is located, which contains several direct and inverted repeats, and a nucleotide stretch that resembles the nicking site of the pC194 family of rolling circle replicating plasmids. Additional binding studies revealed that OrfB binds to an 8 bp inverted repeat that occurs three times within the hairpin structure. The data presented show that OrfB is the replication initiator (Rep) protein of pMEA300, and is therefore termed RepAM. Surprisingly, RepAM lacks significant sequence similarity with known prokaryotic Rep proteins, but it is highly similar to a number of yet uncharacterized ORFs that are located on integrative and conjugative elements of other actinomycetes. It is concluded that RepAM and its homologues are members of a novel class of Rep proteins.
Significant passive protective effect against anthrax by antibody to Bacillus anthracis inactivated spores that lack two virulence plasmids
Authors: Enkhtuya Jargalsaikhan , Kawamoto Keiko , Kobayashi Yoshiyasu , Uchida Ikuo , Rana Neeraj , Makino Sou-ichi
Published: September 2006

The protective-antigen (PA)-based cell-free vaccine is the only vaccine licensed for use against Bacillus anthracis infection in humans. Although the PA shows strong immunogenicity, the capsule or spore-associated somatic antigens may be important as additional vaccine targets for full protection against anthrax. In this study, the protective effect of spore-associated antigens against B. anthracis infection was determined. Rabbits were immunized with formalin-fixed spores of a non-toxigenic unencapsulated B. anthracis strain that lacked the two virulence plasmids pXO1 and pXO2, and the protective effects of the immune antibody were evaluated. Immunostaining and Western blot analysis revealed that the anti-B. anthracis (anti-BA)-spore IgG specifically bound to the surface of spores or endospores of B. anthracis, but not to vegetative cells, or closely related Bacillus species, such as Bacillus cereus, Bacillus subtilis and Bacillus thuringiensis. Passively transferred anti-BA-spore IgG protected mice from intraperitoneal challenge with a lethal dose of fully virulent B. anthracis spores, and increased the survival rate in a dose-dependent manner. Pre-incubation of spores with antibody also reduced their infectivity in a dose-dependent manner. The number of bacteria (c.f.u.) in spleens and livers of infected mice was significantly lower in antibody-treated mice than in untreated mice. Treatment with anti-BA-spore IgG also inhibited the germination of spores in J774.1 macrophages, suggesting that opsonization of spores promotes phagocytosis and subsequent killing by macrophages. These results indicate the usefulness of spore surface antigens as vaccine targets. In combination with major virulence factors such as the PA, spore-associated antigens may offer a safer and more effective multicomponent vaccine for B. anthracis infection.
Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae
Authors: Laue Heike , Schenk Alexander , Li Hongqiao , Lambertsen Lotte , Neu Thomas R. , Molin Søren , Ullrich Matthias S.
Published: September 2006

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.
Complementation of daptomycin dptA and dptD deletion mutations in trans and production of hybrid lipopeptide antibiotics
Authors: Gal Marie-Françoise Coëffet-Le , Thurston Lisa , Rich Paul , Miao Vivian , Baltz Richard H.
Published: September 2006

Daptomycin is a lipopeptide antibiotic produced by Streptomyces roseosporus and recently commercialized as Cubicin® (daptomycin-for-injection) for treatment of skin and skin-structure infections caused by Gram-positive pathogens. Daptomycin is synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by three overlapping genes, dptA, dptBC and dptD. The dptE and dptF genes, immediately upstream of dptA, are likely to be involved in the initiation of daptomycin biosynthesis by coupling decanoic acid to the N-terminal Trp. Analysis of RT-PCR data suggests that dptE, dptF, dptA, dptBC, dptD and possibly other dpt genes are transcribed as one large message; however, it has been demonstrated that sequential translation of these genes from a long transcript is not essential for robust daptomycin production. The dptA and the dptD genes were deleted from the dpt gene cluster, and expressed from ectopic positions in the chromosome under the control of the strong constitutive ermEp* promoter to produce high levels of lipopeptides. This three-locus trans-complementation system was used to produce hybrid lipopeptide antibiotics by introducing the heterologous lptD and cdaPS3 genes from Streptomyces fradiae and Streptomyces coelicolor, respectively, to complement the ΔdptD mutation.
The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin
Authors: Miyamoto Masahiko , Onozato Naohiko , Selvakumar Dakshnamurthy , Kimura Tetsuya , Furuichi Yasuhiro , Komiyama Tadazumi
Published: September 2006

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Δ : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-β-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-β-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-β-glucan synthase.
Identification and characterization of Pseudomonas membrane transporters necessary for utilization of the siderophore pyridine-2,6-bis(thiocarboxylic acid) (PDTC)
Authors: Leach Lynne H. , Lewis Thomas A.
Published: September 2006

The compound pyridine-2,6-bis(thiocarboxylic acid) (PDTC) is known to be produced and excreted by three strains of Pseudomonas. Its reactivity includes the complete dechlorination of the environmental contaminant carbon tetrachloride. PDTC functions as a siderophore; however, roles as a ferric reductant and antimicrobial agent have also been proposed. PDTC function and regulation were further explored by characterizing the phenotypes of mutants in predicted membrane transporter genes. The functions of a predicted outer-membrane transporter (PdtK) and a predicted inner-membrane permease (PdtE) were examined in Pseudomonas putida DSM 3601. Uptake of iron from 55Fe(III):PDTC, and bioutilization of PDTC in a chelated medium, were dependent upon PdtK and PdtE. Another strain of P. putida (KT2440), which lacks pdt orthologues, showed growth inhibition by PDTC that could be relieved by introducing a plasmid containing pdtKCPE. Transcriptional activation in response to exogenously added PDTC (25 μM) was unaltered by the pdtK or pdtE mutations; each mutant showed activation of a pdt transcriptional reporter, indistinguishable from an isogenic PDTC utilization-proficient strain. The data demonstrate that PdtK and PdtE constitute a bipartite outer-membrane/inner-membrane transport system for iron acquisition from Fe(III):PDTC. Disruptions in this portion of the P. putida DSM 3601 pdt gene cluster do not abolish PDTC-dependent transcriptional signalling.
Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103
Authors: Charoenpanich Jittima , Tani Akio , Moriwaki Naoko , Kimbara Kazuhide , Kawai Fusako
Published: September 2006

The genes for polyethylene glycol (PEG) catabolism (pegB,C,D,AandE) inSphingopyxis macrogoltabidastrain 103 were shown to form a PEG-inducible operon. ThepegRgene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putativeσ70-type promoter sites were identified in thepegB,pegAandpegRpromoters. A promoter activity assay showed that thepegBpromoter was induced by PEG and oligomeric ethylene glycols, whereas thepegAandpegRpromoters were induced by PEG. Deletion analysis of thepegBpromoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of thepegBCDAEoperon. Gel retardation assays demonstrated the specific binding of PegR to thepegBpromoter. Transcriptional fusion studies ofpegRwithpegAandpegBpromoters suggested that PegR regulates the expression of thepegBCDAEoperon positively through its binding to thepegBpromoter, but PegR does not bind to thepegApromoter. Two specific binding proteins for thepegApromoter were purified and identified as a GalR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GalR/LacI-type regulator was found in thepegAandpegRpromoters. These results suggested the dual regulation of thepegBCDAEoperon through thepegBpromoter by an AraC-type regulator, PegR (PEG-independent), and through thepegAandpegRpromoters by a GalR/LacI-type regulator together with HU (PEG-dependent).
Functional analysis of 11 putative essential genes in Bacillus subtilis
Authors: Hunt Alison , Rawlins Joy P. , Thomaides Helena B. , Errington Jeff
Published: September 2006

Systematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these (yacA, ydiB, ydiC, ykqC, ylaN, yloQ, ymdA, yneS, yqeI, yqjK and ywlC) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes (ydiB, yloQ, yqeI and ywlC) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.
The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway
Authors: Luong Thanh T. , Lee Chia Y.
Published: September 2006

Most clinical Staphylococcus aureus strains produce either type 5 or type 8 capsular polysaccharides. The production of these capsules is influenced by various environmental factors. To study the regulation of capsule, Tn551 transposon mutagenesis and transcriptional reporter gene fusion were employed to identify several putative regulatory loci that influenced capsule gene expression. One of these, the arl locus, was chosen for further analysis. Tn551 was found to insert within the coding region (near the translational start site of the arlR gene). ArlR, along with ArlS, forms a two-component system that has been previously shown to affect autolysis and production of several secreted proteins. Phenotypic analyses of the arlR-specific mutant and gene fusion analyses showed that arlR activated capsule production at the transcriptional level. However, gel mobility shift assays did not support activation of the capsule genes by direct ArlR binding to the primary cap5 promoter region upstream of the operon. In contrast, it was found that arl activated mgrA, an activator for capsule production, whereas mgrA did not have a significant effect on arlR. Genetic studies supported the notion that arlR functions upstream of mgrA with respect to the regulation of capsule production, although gene fusion studies indicated that arl could also regulate capsule independently from mgrA. Collectively, the results suggest that arl positively regulates capsule production at the transcriptional level primarily through an mgrA-dependent pathway.
Amino acids mediate colony and cell differentiation in the fungal pathogen Candida parapsilosis
Authors: Kim Seong-Kyoun , Bissati Kamal El , Mamoun Choukri Ben
Published: September 2006

Candida parapsilosis is responsible for severe cases of non-albicans systemic candidiasis and is one of the leading causes of mortality in neonates. The molecular mechanisms underlying this organism's virulence remain unknown. Unlike C. albicans, which can exist in several morphogenetic forms, C. parapsilosis exists in either the yeast or pseudohyphal forms. The environmental signals that trigger pseudohyphal differentiation and the signalling pathways that transduce these signals are unknown. This paper provides evidence for the role of amino acids in morphogenesis in C. parapsilosis. The cell and colony morphologies, pseudohyphal differentiation and invasive growth of five C. parapsilosis isolates were characterized in ammonium-rich minimal media lacking or supplemented with naturally occurring amino acids. C. parapsilosis underwent dramatic changes in cellular and colony morphology and formed pseudohyphae in response to a specific subset of amino acids. Transport studies showed that these amino acid inducers activate the transport of some, but not all, unrelated amino acids. Interestingly, citrulline, an amino acid that is not transported in the presence of ammonium, strongly induced pseudohyphal morphogenesis in C. parapsilosis under these conditions. Together the data suggest that amino acids are important morphogens in C. parapsilosis and that amino-acid-mediated morphogenesis in this organism does not require transport of the ligand across the plasma membrane.
Revision of the Agathidinae (Hymenoptera: Braconidae) with comparisons of static and dynamic alignments
Authors: Sharkey Michael J. , Laurenne Nina M. , Sharanowski Barbara , Quicke Donald L. J. , Murray Debra
Published: September 2006

The phylogeny of the Agathidinae (Insecta: Hymenoptera: Braconidae) is investigated based on morphological and sequence data from the D2–3 regions of 28S rDNA. Morphology and molecular data were run simultaneously and separately and the molecular and combined data sets were analyzed using both static, Clustal W, and dynamic, POY, alignments. Both alignments were conducted under a variety of gap costs and results are compared. Sixty‐two ingroup exemplars representing 22 genera and six outgroup taxa representing two subfamilies and five genera were included. Numerous taxa at the generic and tribal levels were tested for monophyly and the evolutionary history of several characters is discussed. The tribe Agathidini s.s. is found to be a derived member of the Microdini and the two are synonymized under the older name, Agathidini s.l. Support is substantial for the tribes Cremnoptini and Disophrini and Earinini but equivocal for the Agathidini s.l. At the generic level, Bassus is found to be polyphyletic. Numerous new synonymies and combinations are proposed.

© The Willi Hennig Society 2006.
Re‐organization of the cytoskeleton and endoplasmic reticulum in the Arabidopsis pen1‐1 mutant inoculated with the non‐adapted powdery mildew pathogen, Blumeria graminis f. sp. hordei
Authors: TAKEMOTO DAIGO , JONES DAVID A. , HARDHAM ADRIENNE R.
Published: September 2006

Plant cells attacked by microorganisms rapidly translocate cytoplasm to the site of pathogen penetration, a response that usually involves rearrangement of actin microfilaments. In this study, we monitored re‐organization of green fluorescent protein (GFP)‐labelled actin microfilaments, microtubules and endoplasmic reticulum (ER) during infection by the powdery mildew pathogen Blumeria graminis f. sp. hordei in non‐host Arabidopsis and in the Arabidopsis penetration 1‐1 (pen1‐1) mutant, which shows increased penetration susceptibility to non‐adapted pathogens. Comparison of pen1‐1 with wild‐type Arabidopsis showed that the actin, microtubule and ER networks all responded in the pen1‐1 mutant as they do in wild‐type plants. Actin microfilaments became focused on the penetration site and ER accumulated at the penetration site while the overall arrangement of microtubule arrays was largely unaffected. These results indicate that the block in vesicle secretion conferred by the pen1‐1 mutation does not interfere with cytoplasmic aggregation or recruitment of actin or ER to the infection site. In the pen1‐1 mutant, the higher rate of successful penetration by the non‐adapted pathogen results in an increased incidence of hypersensitive cell death. In dying cells, the structure of the ER was rapidly destroyed, in contrast to actin microfilaments and microtubules which remained for a longer time after the initiation of cell death. In Arabidopsis with GFP‐tagged tubulin, fluorescent vesicle‐like structures appeared near the cell surface during the initiation of cell death. In both wild‐type and pen1‐1 mutant plants, in cells surrounding the dying cell, bundles of actin microfilaments focused on the anticlinal walls adjacent to the dead cell and ER accumulated in the cortical cytoplasm near the dead cell. These observations suggest that the neighbouring, non‐infected cells use actin‐based transport to secrete material at the surface adjacent to the dead cell. They also indicate that disruption of secretion (in the pen1‐1 mutant) does not inhibit transmission of signals from the dying cells to their neighbours.
A novel link between tomato GRAS genes, plant disease resistance and mechanical stress response
Authors: MAYROSE MAYA , EKENGREN SOPHIA K. , MELECH‐BONFIL SHIRI , MARTIN GREGORY B. , SESSA GUIDO
Published: September 2006

Members of the GRAS family of transcriptional regulators have been implicated in the control of plant growth and development, and in the interaction of plants with symbiotic bacteria. Here we examine the complexity of the GRAS gene family in tomato (Solanum lycopersicum) and investigate its role in disease resistance and mechanical stress. A large number of tomato ESTs corresponding to GRAS transcripts were retrieved from the public database and assembled in 17 contigs of putative genes. Expression analysis of these genes by real‐time RT‐PCR revealed that six SlGRAS transcripts accumulate during the onset of disease resistance to Pseudomonas syringae pv. tomato. Further analysis of two selected family members showed that their transcripts preferentially accumulate in tomato plants in response to different avirulent bacteria or to the fungal elicitor EIX, and their expression kinetics correlate with the appearance of the hypersensitive response. In addition, transcript levels of eight SlGRAS genes, including all the Pseudomonas‐inducible family members, increased in response to mechanical stress much earlier than upon pathogen attack. Accumulation of SlGRAS transcripts following mechanical stress was in part dependent on the signalling molecule jasmonic acid. Remarkably, suppression of SlGRAS6 gene expression by virus‐induced gene silencing impaired tomato resistance to P. syringae pv. tomato. These results support a function for GRAS transcriptional regulators in the plant response to biotic and abiotic stress.
Quantitative measurement of hepatitis C virus core antigen is affected by the presence of cryoglobulins
Authors: Pham B-N , Martinot-Peignoux M , Ripault M-P , Boyer N , Levy V , Marcellin P
Published: September 2006

Mixed cryoglobulinaemia is associated strikingly with HCV infection. The aim of this study was to assess whether the adherence to proper methods of collecting samples for cryoglobulin detection was critical or not on virological parameters in hepatitis C virus (HCV) patients. We studied 56 consecutive patients. Blood samples were collected using a conventional method and a blood collection method at 37°C adapted to cryoglobulin detection. HCV core antigen and HCV RNA were measured in sera and cryoglobulins issued from both blood collection methods. In cryoglobulin-positive patients, serum concentrations of HCV core antigen, but not that of HCV RNA, were significantly higher when a conventional method was used, compared to a blood collection method at 37°C (P = 0·001). In the cryoprecipitates, concentration of HCV core antigen was optimum when the blood collection method at 37°C, rather than the conventional method, was applied for cryoglobulin detection (P < 10−4). The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37°C rather than the conventional method (P < 0·001). HCV parameter measurements and cryoglobulin study should not be performed on the same serum samples due to the potential impact of blood collection methods on results.
Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-β expression in the joint
Authors: Marinova-Mutafchieva L , Gabay C , Funa K , Williams R O
Published: September 2006

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self-limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro- and anti-inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6] and anti-inflammatory cytokines [IL-10, IL-1ra, transforming growth factor (TGF)-β1, TGF-β2 and TGF-β3] in joints was carried out over the course of the disease. Both pro- and anti-inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti-inflammatory cytokines, particularly TGF-β1 and TGF-β2, increased. Surprisingly, TNF-α continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF-α during the period of disease remission had no effect on TGF-β expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro- and anti-inflammatory cytokine expression in the joints. Of the anti-inflammatory cytokines studied, TGF-β1 and TGF-β2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.
Phenotypic features of circulating leucocytes as immunological markers for clinical status and bone marrow parasite density in dogs naturally infected by Leishmania chagasi
Authors: Reis A B , Teixeira-Carvalho A , Giunchetti R C , Guerra L L , Carvalho M G , Mayrink W , Genaro O , Corrêa-Oliveira R , Martins-Filho O A
Published: September 2006

Canine visceral leishmaniasis (CVL) manifests itself as a broad clinical spectrum ranging from asymptomatic infection to patent severe disease. Despite relevant findings suggesting changes on lymphocytes subsets regarding the CVL clinical forms, it still remains to be elucidated whether a distinct phenotypic profile would be correlated with degree of tissue parasite density. Herein, we have assessed the correlation between the clinical status as well as the impact of bone marrow parasite density on the phenotypic profile of peripheral blood leucocytes in 40 Brazilian dogs naturally infected by Leishmania chagasi. Our major findings describe the lower frequency of B cells and monocytes as the most important markers of severe CVL. Our main statistically significant findings reveal that the CD8+ T cell subset reflects most accurately both the clinical status and the overall bone marrow parasite density, as increased levels of CD8+ lymphocytes appeared as the major phenotypic feature of asymptomatic disease and dogs bearing a low parasite load. Moreover, enhanced major histocompatibility complex (MHC)-II density as well as a higher CD45RB/CD45RA expression index seems to represent a key element to control disease morbidity. The association between clinical status, bone marrow parasitism and CD8+ T cells re-emphasizes the role of the T cell-mediated immune response in the resistance mechanisms during ongoing CVL. Higher levels of circulating T lymphocytes (both CD4+ and CD8+ T cells) and lower MHC-II expression by peripheral blood lymphocytes seem to be the key for the effective immunological response, a hallmark of asymptomatic CVL.
Alcohol-induced gastritis prevents oral tolerance induction in mice
Authors: Andrade M C , Menezes J S , Cassali G D , Martins-Filho O A , Cara D C , Faria A M C
Published: September 2006

Despite several reports on the immunological relationship between inflammatory bowel diseases and immunoregulatory mechanisms in the gut, systematic studies addressing the impact of inflammatory processes in the gastric mucosa on events, such as oral tolerance, are still limited. Herein, we report the establishment of a novel murine model of gastritis induced by short-term administration of ethanol. The major immumological features of this clinical entity are characterized, as well as its impact on the induction of oral tolerance. Our data demonstrate that ethanol ingestion during 4 consecutive days triggered an acute inflammatory reaction in the stomach referred as ethanol-induced gastritis and characterized by hyperaemia, oedema and mixed mononuclear/polymorphonuclear cell infiltrate. Besides local immunological changes, such as high levels of gastric interleukin (IL)-4 and interferon (IFN)-γ, systemic alterations are also observed, including increased IL-4 synthesis, enhanced levels of serum IgE and absence of IL-10 production by spleen cells. Moreover, ethanol-induced gastritis prevents oral tolerance induction to ovalbumin (OVA) as demonstrated by unaltered anti-OVA humoral and cellular immune responses in treated animals. Tissue eosinophilia after footpad immunization with OVA suggests that oral treatment with ethanol induced an allergic-type reaction. Taken together, our findings indicate that short-term ethanol ingestion is associated with gastric inflammatory events able to break immunoregulatory mechanisms that maintain mucosal homeostasis and oral tolerance.
The myeloid differentiation factor 88 is dispensable for the development of a delayed host response to Pseudomonas aeruginosa lung infection in mice
Authors: Power M R , Marshall J S , Yamamoto M , Akira S , Lin T-J
Published: September 2006

Because MyD88 transduces a core set of Toll-like receptor (TLR)-induced signals, microbial-induced host responses can be divided broadly into the MyD88-dependent and MyD88-independent pathways. A specific pathogen induces a distinct pattern of host response dependent upon the signalling pathways employed. Recently, we demonstrated that a MyD88-dependent pathway is essential for the development of early (4–8 h) host response to Pseudomonas aeruginosa lung infection. Here, we show that the development of a delayed (24–48 h) host response to P. aeruginosa is independent of MyD88. Using MyD88-deficient mice, the production of macrophage inflammatory protein 2, tumour necrosis factor and interleukin 1α in the airway was observed following P. aeruginosa lung infection for 24 or 48 h. Moreover, the MyD88-deficient mice recruited sufficient neutrophils in the lung and cleared the bacteria efficiently from the lung after 48 h. Thus, the full development of host responses to P. aeruginosa lung infection involves, in a sequential, stepwise fashion, a MyD88-dependent early response and a MyD88-independent delayed mechanism.
Expression and function of the purinergic receptor P2X7 in patients with pulmonary tuberculosis
Authors: Franco-Martínez S , Niño-Moreno P , Bernal-Silva S , Baranda L , Rocha-Meza M , Portales-Cervantes L , Layseca-Espinosa E , González-Amaro R , Portales-Pérez D
Published: September 2006

P2X7 is a channel receptor gated by adenosine triphosphate (ATP) that is involved in the killing of intracellular mycobacteria. To explore further the role of P2X7 in immunity against Mycobacterium tuberculosis, we studied its expression and function in 19 patients with pulmonary tuberculosis (TB) and 19 healthy contacts. Flow cytometry analysis showed a similar and variable expression of P2X7 in TB patients and healthy subjects. In contrast, P2X7 mARN levels were significantly higher in TB patients. When the function of the P2X7 receptor in peripheral blood mononuclear cells (PBMC) was assessed by the effect of exogenous ATP on apoptosis, the uptake of the fluorescent marker Lucifer yellow or extracellular signal regulated kinase (ERK) phosphorylation, no significant differences were detected in patients and controls. However, mRNA macroarray analysis showed that upon stimulation with ATP, the PBMC from TB patients showed a significant induction of a higher number of cytokine genes (27 of 96), and a lower number of apoptosis genes (20 of 96) compared to healthy controls (17 and 76 genes, respectively). These results suggest that although the PBMC from TB patients do not show apparent abnormalities in the expression of P2X7, and the intracellular signals generated through it, the pattern of gene expression induced by ATP in these cells is different from that found in healthy contacts. This phenomenon suggests a defective function of P2X7 in the immune cells from TB patients, a condition that may contribute to the inability of these patients to eliminate the mycobacteria.
Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice
Authors: Ito R , Shin-Ya M , Kishida T , Urano A , Takada R , Sakagami J , Imanishi J , Kita M , Ueda Y , Iwakura Y , Kataoka K , Okanoue T , Mazda O
Published: September 2006

Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-γ, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-γ–/– and wild-type (WT) C57BL/6 mice were fed 2·5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-γ in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-γ deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-γ–/– mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-γ (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-γ–/– mice. The present results demonstrate clearly that IFN-γ plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-γ-dependent fashion.
Autoantibodies against CD28 are associated with atopic diseases
Authors: Neuber K , Mähnss B , Hübner C , Gergely H , Weichenthal M
Published: September 2006

The B7-1/B7-2-CD28/CTLA-4 pathway is crucial in regulating T cell activation and tolerance. Autoantibodies to surface molecules on lymphocytes have already been described in various immune conditions, such as autoimmune diseases, infections and blood transfusions. The objective of this study was to test sera from healthy individuals and from patients for association of CD28 autoantibodies with inflammatory and non-inflammatory diseases. First, CD28 was obtained by digestion of CD28-Ig fusion protein with trypsin. The cleavage products were separated by sodium dodecyl sulphate–page gel electrophoresis. Additionally, a CD28/GST fusion protein was expressed in Escherichia coli and was used to establish an enzyme-linked immunosorbent assay for detection of autoantibodies against CD28. Sera from healthy individuals (n = 72) and patients with different inflammatory and non-inflammatory skin diseases (n = 196) were tested for the presence of autoantibodies against CD28. Using mixed lymphocyte reaction (MLR), purified autoantibodies against CD28 were tested for their effects on CTLA-4-Ig-induced T cell anergy. In this study, for the first time, we describe the existence of autoantibodies against CD28 in humans which are associated with atopic diseases, e.g. allergic rhinitis and asthma. These antibodies stimulate T cells and overcome the CTLA-4-Ig-induced anergy of T cells in an MLR. The existence of autoantibodies against CD28, which may have a T cell-stimulating function, has been shown. The data indicate that autoantibodies against CD28 could be a new immunological mechanism in allergic inflammation. Additionally, autoantibodies against CD28 could be an important new marker to discriminate between atopic diseases and other inflammatory skin diseases.
Rapid climate change‐related growth decline at the southern range edge of Fagus sylvatica
Authors: JUMP ALISTAIR S. , HUNT JENNY M. , PEÑUELAS JOSEP
Published: September 2006

Studies on Fagus sylvatica show that growth in populations toward the southern limit of this species' distribution is limited strongly by drought. Warming temperatures in the Mediterranean region are expected to exacerbate drought where they are not accompanied by increases in precipitation. We studied levels of annual growth in mature F. sylvatica trees over the last half‐century in the Montseny Mountains in Catalonia (northeast Spain). Our results show significantly lower growth of mature trees at the lower limit of this species' distribution when compared with trees at higher altitudes. Growth at the lower Fagus limit is characterized by a rapid recent decline starting in approximately 1975. By 2003, growth of mature trees had fallen by 49% when compared with predecline levels. This is not an age‐related phenomenon, nor is it seen in comparable populations at higher altitudes. Analysis of climate‐growth relationships suggests that the observed decline in growth is a result of warming temperatures and that, as precipitation in the region has not increased, precipitation is now insufficient to ameliorate the negative effects of increased temperatures on tree growth. As the climate‐response of the studied forest is comparable with that of F. sylvatica forests in other southern European regions, it is possible that this growth decline is a more widespread phenomenon. Warming temperatures may lead to a rapid decline in the growth of range‐edge populations and a consequent retreat of the species distribution in southern Europe. Assessment of long‐term growth trends across the southern range edge of F. sylvatica therefore merits further attention.
The interdependence of fire, grass, kangaroos and Australian Aborigines: a case study from central Arnhem Land, northern Australia
Authors: Murphy Brett P. , Bowman David M. J. S.
Published: September 2006

Aim  To describe the nexus between Aboriginal landscape burning and patterns of habitat use by kangaroos in a tropical savanna habitat mosaic, and to provide evidence to evaluate the claim that Aboriginal landscape burning is a game management tool.

Location  Central Arnhem Land, a stronghold of traditional Aboriginal culture, in the monsoon tropics of northern Australia.

Methods  The abundance of kangaroo scats was recorded throughout a landscape burnt by Aboriginal people, and used as a proxy for the intensity of habitat use by kangaroos. Scat abundance was assessed along field traverses totalling 112 km, at three time periods: (1) 1–4 weeks following mid‐dry season burning (July 2003); (2) in the late dry season (November 2003); and (3) in the following mid‐dry season (July 2004). We compared the intensity with which kangaroos used burnt vs. unburnt areas in various habitat types, with time since mid‐dry season burning. Scats were collected from areas that had been burnt to a varying extent and the abundance of carbon and nitrogen stable isotopes (δ13C and δ15N) and carbon to nitrogen ratios (C : N) determined.

Results  There was clear evidence of an interaction between burning and habitat type on the abundance of kangaroo scats. Scats were much more abundant in burnt vs. unburnt areas in the moist habitats, but the opposite effect was observed in the dry rocky habitats, with higher scat abundance in unburnt areas. This interactive effect of burning and habitat type on scat abundance was observed immediately (< 4 weeks) following fire, and was still present one year later. High concentrations of nitrogen in resprouting grasses indicate that burnt areas may provide kangaroos with greater access to nutrients. The isotopic composition of scats indicates that kangaroos feeding in extensively burnt areas were consuming more grasses, and possibly sedges, than kangaroos feeding in unburnt areas.

Main conclusions  The fine‐scale mosaic of burnt and unburnt areas created by mid‐dry season Aboriginal landscape burning has clear effects on the distribution of kangaroos. Kangaroos move into burnt moist habitats and away from burnt dry, rocky habitats. Isotopic analysis of scats suggests that the mechanism driving this effect is the increased abundance of nitrogen rich grasses in burnt moist habitats.