Transcriptomic characterization of two major Fusarium resistance quantitative trait loci (QTLs), Fhb1 and Qfhs.ifa-5A, identifies novel candidate genes

Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248

Electroexcitation of the Roper resonance from CLAS data

The helicity amplitudes of the electroexcitation of the Roper resonance on proton are extracted at 1.7 < Q2 < 4.2 GeV2 from recent high precision JLab-CLAS cross sections data and longitudinally polarized beam asymmetry for pi+ electroproduction on protons. The analysis is made using two approaches, dispersion relations and unitary isobar model, which give consistent results. It is found that the transverse helicity amplitude for the gamma* p --> P11(1440) transition, which is large and negative at Q2=0, becomes large and positive at Q2 ~ 2 GeV2, and then drops slowly with Q2. Longitudinal helicity amplitude, that was previously found from CLAS data as large and positive at Q2=0.4,0.65 GeV2, drops with Q2. These results rule out the presentation of P11(1440) as a 3qG hybrid state, and provide strong evidence in favor of this resonance as a first radial excitation of the 3q ground state.Comment: 3 pages, 2 figures, Talk on the Workshop on "The Physics of Excited Nucleons", Bonn, Germany, October 200

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results

Aplicación de parámetros farmacocinéticos/farmacodinámicos al empleo terapéutico de amoxicilina en gatos domésticos

La amoxicilina (AMX) es un antibiótico betalactámico que incluye en su espectro microorganismos grampositivos (estafilococos y estreptococos) y gramnegativos (algunas enterobacterias). La AMX puede administrarse por vía parenteral como formulaciones solubles de liberación y acción rápida o, como formulaciones de liberación sostenida. Los intervalos posológicos habitualmente recomendados son cada 12 h para las formulaciones solubles y, cada 48 h para las formulaciones de depósito. La eficacia clínica de la AMX es "tiempo dependiente" y el indicador empleado para predecir el éxito terapéutico es el T>CIM (tiempo que las concentraciones plasmáticas se encuentran por encima de la CIM del patógeno a tratar). El éxito terapéutico se obtendría con un T CIM ≥40% del intervalo posológico. El propósito de este trabajo es determinar el T CIM para la AMX luego de su administración intravenosa (IV), intramuscular en forma soluble (IMs) e, intramuscular como suspensión de depósito (IMd) a gatos domésticos.Facultad de Ciencias Veterinaria

Aplicación de parámetros farmacocinéticos/farmacodinámicos al empleo terapéutico de amoxicilina en gatos domésticos

La amoxicilina (AMX) es un antibiótico betalactámico que incluye en su espectro microorganismos grampositivos (estafilococos y estreptococos) y gramnegativos (algunas enterobacterias). La AMX puede administrarse por vía parenteral como formulaciones solubles de liberación y acción rápida o, como formulaciones de liberación sostenida. Los intervalos posológicos habitualmente recomendados son cada 12 h para las formulaciones solubles y, cada 48 h para las formulaciones de depósito. La eficacia clínica de la AMX es "tiempo dependiente" y el indicador empleado para predecir el éxito terapéutico es el T>CIM (tiempo que las concentraciones plasmáticas se encuentran por encima de la CIM del patógeno a tratar). El éxito terapéutico se obtendría con un T CIM ≥40% del intervalo posológico. El propósito de este trabajo es determinar el T CIM para la AMX luego de su administración intravenosa (IV), intramuscular en forma soluble (IMs) e, intramuscular como suspensión de depósito (IMd) a gatos domésticos.Facultad de Ciencias Veterinaria

Aplicación de parámetros farmacocinéticos/farmacodinámicos al empleo terapéutico de amoxicilina en gatos domésticos

La amoxicilina (AMX) es un antibiótico betalactámico que incluye en su espectro microorganismos grampositivos (estafilococos y estreptococos) y gramnegativos (algunas enterobacterias). La AMX puede administrarse por vía parenteral como formulaciones solubles de liberación y acción rápida o, como formulaciones de liberación sostenida. Los intervalos posológicos habitualmente recomendados son cada 12 h para las formulaciones solubles y, cada 48 h para las formulaciones de depósito. La eficacia clínica de la AMX es "tiempo dependiente" y el indicador empleado para predecir el éxito terapéutico es el T>CIM (tiempo que las concentraciones plasmáticas se encuentran por encima de la CIM del patógeno a tratar). El éxito terapéutico se obtendría con un T CIM ≥40% del intervalo posológico. El propósito de este trabajo es determinar el T CIM para la AMX luego de su administración intravenosa (IV), intramuscular en forma soluble (IMs) e, intramuscular como suspensión de depósito (IMd) a gatos domésticos.Facultad de Ciencias Veterinaria

Transport of Proteins into Mitochondria

The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120000 and 500000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200000–400000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20–30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form its precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein

Genomic and transcriptional analysis of protein heterogeneity of the honeybee venom allergen Api m 6

Several components of honeybee venom are known to cause allergenic responses in humans and other vertebrates. One such component, the minor allergen Api m 6, has been known to show amino acid variation but the genetic mechanism for this variation is unknown. Here we show that Api m 6 is derived from a single locus, and that substantial protein-level variation has a simple genome-level cause, without the need to invoke multiple loci or alternatively spliced exons. Api m 6 sits near a misassembled section of the honeybee genome sequence, and we propose that a substantial number of indels at and near Api m 6 might be the root cause of this misassembly. We suggest that genes such as Api m 6 with coding-region or untranslated region indels might have had a strong effect on the assembly of this draft of the honeybee genome