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991.
Bork A. Berghoff Jens Glaeser Cynthia M. Sharma Jörg Vogel Gabriele Klug 《Molecular microbiology》2009,74(6):1497-1512
Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (1O2) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to 1O2. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with 1O2 or superoxide (O–2). One sRNA was specifically induced by 1O2 and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by 1O2 and O–2 were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoHI and RpoHII. The most abundant sRNA was processed in the presence of 1O2 but not by O–2. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of 1O2‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by 1O2 exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress. 相似文献
992.
Tilsner J Linnik O Christensen NM Bell K Roberts IM Lacomme C Oparka KJ 《The Plant journal : for cell and molecular biology》2009,57(4):758-770
We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed. 相似文献
993.
Anna Karlgren Jenny Carlsson Niclas Gyllenstrand Ulf Lagercrantz Jens F. Sundstr?m 《Journal of visualized experiments : JoVE》2009,(26)
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.Anna Karlgren and Jenny Carlsson contributed equally to this study.Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se 相似文献
994.
Ute Achenbach Joao Paulo Evgenyia Ilarionova Jens Lübeck Josef Strahwald Eckhard Tacke Hans-Reinhard Hofferbert Christiane Gebhardt 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,118(3):619-629
The damage caused by the parasitic root cyst nematode Globodera pallida is a major yield-limiting factor in potato cultivation . Breeding for resistance is facilitated by the PCR-based marker ‘HC’, which is diagnostic for an allele conferring high resistance
against G. pallida pathotype Pa2/3 that has been introgressed from the wild potato species Solanum vernei into the Solanum tuberosum tetraploid breeding pool. The major quantitative trait locus (QTL) controlling this nematode resistance maps on potato chromosome
V in a hot spot for resistance to various pathogens including nematodes and the oomycete Phytophthora infestans. An unstructured sample of 79 tetraploid, highly heterozygous varieties and breeding clones was selected based on presence
(41 genotypes) or absence (38 genotypes) of the HC marker. Testing the clones for resistance to G. pallida confirmed the diagnostic power of the HC marker. The 79 individuals were genotyped for 100 single nucleotide polymorphisms
(SNPs) at 10 loci distributed over 38 cM on chromosome V. Forty-five SNPs at six loci spanning 2 cM in the interval between
markers GP21-GP179 were associated with resistance to G. pallida. Based on linkage disequilibrium (LD) between SNP markers, six LD groups comprising between 2 and 18 SNPs were identified.
The LD groups indicated the existence of multiple alleles at a single resistance locus or at several, physically linked resistance
loci. LD group C comprising 18 SNPs corresponded to the ‘HC’ marker. LD group E included 16 SNPs and showed an association
peak, which positioned one nematode resistance locus physically close to the R1 gene family.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
995.
Salma Nowroozalizadeh Fredrik Mnsson Zacarias da Silva Johanna Repits Braima Dabo Carla Pereira Antonio Biague Jan Albert Jens Nielsen Peter Aaby Eva Maria Feny Hans Norrgren Birgitta Holmgren Marianne Jansson 《Cytokine》2009,46(3):325-331
Background: HIV-1 and HIV-2 are two related viruses with distinct clinical outcomes, where HIV-1 is more pathogenic and transmissible than HIV-2. The pathogenesis of both infections is influenced by the dysregulation and deterioration of the adaptive immune system. However, their effects on the responsiveness of innate immunity are less well known. Here, we report on toll-like receptor (TLR) stimuli responsiveness in HIV-1 or HIV-2 infections. Methods: Whole blood from 235 individuals living in Guinea-Bissau who were uninfected, infected with HIV-1, infected with HIV-2, and/or infected with HTLV-I, was stimulated with TLR7/8 and TLR9 agonists, R-848 and unmethylated CpG DNA. After TLR7/8 and TLR9 stimuli, the expression levels of IL-12 and IFN-α were related to gender, age, infection status, CD4+ T cell counts, and plasma viral load. Results: Defective TLR9 responsiveness was observed in the advanced disease stage, along with CD4+ T cell loss in both HIV-1 and HIV-2 infections. Moreover, TLR7/8 responsiveness was reduced in HIV-1 infected individuals compared with uninfected controls. Conclusions: Innate immunity responsiveness can be monitored by whole blood stimulation. Both advanced HIV-1 and HIV-2 infections may cause innate immunity dysregulation. 相似文献
996.
Christina Krabbe† Elise Courtois‡ Pia Jensen Jesper R. Jørgensen† Jens Zimmer Alberto Martínez-Serrano‡ Morten Meyer 《Journal of neurochemistry》2009,110(6):1908-1920
Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-xL and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-xL over-expressing subline (hVMbcl-xL ) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-xL cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-xL -over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-xL cell cultures compared with control. We conclude that Bcl-xL and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells. 相似文献
997.
Stephan Reitinger Johannes M��llegger Brigitte Greiderer Jens Erik Nielsen G��nter Lepperdinger 《The Journal of biological chemistry》2009,284(29):19173-19177
Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.Hyaluronan (HA),3 a linear polysaccharide found in the extracellular matrix of most tissues and body fluids of vertebrates, is enzymatically degraded by hyaluronidases (1). Mammalian-type hyaluronidases are grouped into EC 3.2.1.35 (2, 3) or the glycoside hydrolase family 56 (4). Members of this enzyme family hydrolyze the 1,4-β-glycosidic linkage between N-acetyl-d-glucosamine and d-glucuronate within HA polymers (5). In mammalians, hyaluronidases have been found in testis, liver lysosomes, and serum. They are involved in controlling HA levels and are thus implicated in various diseases related to defects of HA metabolism (6).The crystal structures of hyaluronidase from bee (7), wasp (8), and only recently that of human serum hyaluronidase 1 (HYAL1) (9) have been deciphered. In addition to the N-terminal catalytic domain of the insect enzymes, which resembles a distorted (β/α)8 barrel, HYAL1 contains yet another domain. HA hydrolysis is achieved by a pair of acidic amino acids via a retaining double displacement mechanism and a substrate-assisted catalysis, in which the carbonyl oxygen of the N-acetyl group of the cleaved HA subunit acts as the catalytic nucleophile (7).Mammalian-type hyaluronidases display different pH optima. HYAL1 (10) and hyaluronidase 2 (HYAL2) (11) exhibit highest activities at acidic conditions, whereas the hyaluronidase found in Xenopus laevis kidney is only active at neutral pH (12). Bee venom hyaluronidase (13), as well as sperm hyaluronidase, PH20 (SPAM1) (14), are capable of degrading HA over a broad pH range. Up to three PH20 isoforms with greatly different pH optima could be found in protein preparations from bovine testis (15). Extensive analysis of hyaluronidase structures did not bring forward any insights as to what residues or regions of the enzymes specify a specific pH optimum.Profiles of pH-dependent activities can be assigned by computing the electrostatic interactions of the enzyme, which are primarily determined by the ionization states of its amino acid side chains. The pKa values of titratable groups of the enzyme reflect pH-dependent properties such as stability, enzymatic interaction, and substrate interactions (16). Here we present computational and experimental data on the replacement of a loop region located at the end of the substrate binding groove yielding a variant hyaluronidase with an altered pH profile. 相似文献
998.
Jens Lättig Alexander Oksche Michael Beyermann Walter Rosenthal Gerd Krause 《Journal of peptide science》2009,15(7):479-491
The molecular basis for recognition of peptide ligands endothelin‐1, ‐2 and ‐3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ETA or ETB is not clearly resolved. We derived sequence‐structure‐function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10‐fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ETA is restrictive for a selected group of peptide ligands' N‐termini, whereas a broad funnel‐shaped entrance in ETB accepts a variety of different shapes and properties of ligands. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
999.
Stefan U. Weber Andreas Koch Jens Kankeleit Jens-Christian Schewe Ullrich Siekmann Frank Stüber Andreas Hoeft Stefan Schröder 《Apoptosis : an international journal on programmed cell death》2009,14(1):97-107
During therapeutic hyperbaric oxygenation lymphocytes are exposed to high partial pressures of oxygen. This study aimed to
analyze the mechanism of apoptosis induction by hyperbaric oxygen. For intervals of 0.5–4 h Jurkat-T-cells were exposed to
ambient air or oxygen atmospheres at 1–3 absolute atmospheres. Apoptosis was analyzed by phosphatidylserine externalization,
caspase-3 activation and DNA-fragmentation using flow cytometry. Apoptosis was already induced after 30 min of hyperbaric
oxygenation (HBO, P < 0.05). The death receptor Fas was downregulated. Inhibition of caspase-9 but not caspase-8 blocked apoptosis induction
by HBO. Hyperbaric oxygen caused a loss of mitochondrial membrane potential and caspase-9 induction. The mitochondrial pro-survival
protein Bcl-2 was upregulated, and antagonizing Bcl-2 function potentiated apoptosis induction by HBO. In conclusion, a single
exposure to hyperbaric oxygenation induces lymphocyte apoptosis by a mitochondrial and not a Fas-related mechanism. Regulation
of Fas and Bcl-2 may be regarded as protective measures of the cell in response to hyperbaric oxygen. 相似文献
1000.
Anja W. Fjorback Patrick Pla Heidi K. Müller Frédéric Saudou Jens R. Nyengaard 《Biochemical and biophysical research communications》2009,380(4):724-728
The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy. 相似文献