Effects of tubers of the Jerusalem artichoke (Helianthus tuberosus) and potatoes (Solanum tuberosum) on the intestinal microbiota of pigs and evaluation of a procedure for quantification of microbial mass in pig faeces

Die Mikrobiota im Gastrointestinaltrakt (GIT) spielt eine bedeutende Rolle beim Fermentationsprozess im Bezug auf die Nährstoffversorgung sowie die Gesundheit des Darms und des gesamten Organismus. Inulin und resistente Stärke (RS) konnten als präbiotisch wirksame Substanzen identifiziert werden und sind jeweils auch in den Knollen der Topinamburpflanze (Helianthus tuberosus) und in Kartoffeln (Solanum tuberosum) enthalten. Da sie ebenfalls energiereiche Futtermittel für Schweine sind, war es das Ziel der ersten beiden Studien, die Auswirkungen der Aufnahme von Topinamburknollen und Kartoffeln auf die intestinale Mikrobiota und Parameter des Immunsystems bei Endmastschweinen zu bestimmen. In der dritten Studie wurde die mikrobielle Biomasse quantitativ mit einem Verfahren zur Isolation von Bakterien in einer Flüssigkeit durch Hochgeschwindigkeits-Zentrifugation erfasst und der bakteriell gebundene Stickstoff (MP-N) mit dem bakteriellen und endogenem Kotstickstoff (BEDN) verglichen. Im ersten Versuch wurden 72 Endmastschweine in einem Freilandhaltungssystem in eine Kontroll- (CT), die mit Kraftfutter entsprechend des Bedarfs der Tiere für ein Leistungsniveau von 700 g täglichem Lebendmassezuwachs versorgt wurde, und eine Versuchsvariante (ET) aufgeteilt. In der Versuchsvariante erhielten die Tiere nur 70% der Kraftfuttermenge der Kontrollvariante, hatten aber Zugang zu einer abgeteilten Fläche, auf der Topinamburknollen angebaut waren. Die freie Aufnahme von Topinamburknollen wurde auf 1•24 kg Trockenmasse (TM)/Tag bestimmt, entsprechend einer Inulinaufnahme von durchschnittlich 800 g/Tag. Während sich die Wachstumsleistung in der Kontrollvariante auf 0•642 ± 0•014 kg/Tag belief, war sie in der Versuchsvariante mit 0•765 ± 0•015 kg/Tag (P=0•000) höher. Die freie Verfügbarkeit von Inulin und Fructo-oligosacchariden (FOS) im GIT der Schweine erhöhte die Keimzahlen der anaeroben Bakterien (P=0•000), Laktobazillen (P=0•046) und Hefen (P=0•000) signifikant und verringerte das Vorkommen von Clostridium perfringens im Schweinekot erheblich von lg 5•24 ± 0•17 kolonie-bildende Einheiten pro g Frischmasse (KbE/ g FM) in der Kontrollvariante auf lg 0•96 ± 0•20 KbE/ g FM in der Versuchsvariante (P=0•000). C-reaktives Protein (CRP) und Antikörper gegen Lipopolysaccharide (LPS) von Escherichia coli J5 ließen keine Unterschiede zwischen den Fütterungsvarianten erkennen. In der zweiten Untersuchung wurden 58 Endmastschweine einer Kontrollvariante (CT), die bedarfsgerecht mit einer Kraftfuttermischung für ein Leistungsniveau von 700 g Tageszunahmen gefüttert wurde, und zwei Versuchsvarianten zugeteilt. Die Versuchsvarianten erhielten eine Menge von 1•2 kg TM gedämpften Kartoffeln (potato treatment, PT) oder gedämpften und einsilierten Kartoffeln (silage treatment, ST) pro Tag und nur 46% bzw. 43% der Menge des Kraftfutters der Kontrollvariante. Die Wachstumsleistung und Schlachtkörperzusammensetzung ließen keine signifikanten Unterschiede zwischen den Varianten erkennen. Im PT und ST waren gegenüber dem CT im Kot der pH-Wert sowie die Gehalte von TM, Neutral-Detergenz-Faser (NDF), unverdautem Futterstickstoff (UDN) und teilweise von Säure-Detergenz-Faser (ADF) signifikant niedriger (P=0•000) und die von Ammonium (NH4) und Ammoniumstickstoff (NH4-N) signifikant höher (P=0•000). Das hohe Angebot von hitzebehandelten Kartoffeln führte zu einer erheblichen Verringerung von E. coli (P=0•000), C. perfringens (P=0•000) und Immunoglobulin A gegen LPS von E. coli J5 (P=0•001). Darüber hinaus waren in der ersten Versuchsperiode im ST die aeroben und anaeroben Gesamtkeimzahlen sowie die Laktobazillen und Hefen gegenüber dem PT signifikant erhöht. Die Unterschiede in der Mikrobiota zwischen der Kontroll- und Versuchsvarianten weisen auf die positiven Auswirkungen von Topinamburknollen und hitzebehandelten Kartoffeln auf die Mikrobiota im hinteren Darmabschnitt hin. Das Ziel der dritten Untersuchung war die Modifizierung des Verfahrens zur Isolation von Bakterien in einer Flüssigkeit mittels verschiedener Zentrifugationsschritte, um ein mikrobielles Pellet (MP) zu erhalten, welches die quantitative Abtrennung und Erfassung der Bakterien in Schweinekot ermöglicht. Zusätzlich wurde der BEDN Anteil sowie die Gehalte der Aminozucker Galactosamin, Glucosamin, Mannosamin und Muraminsäure im Kot und im MP bestimmt. Die untersuchten Kotproben stammten von Schweinen eines Phosphor (P) Stoffwechselversuch. Zehn männlich-kastrierte Schweine mit einem durchschnittlichen Lebendgewicht von 51•1 ± 8•5 kg wurden einzeln in Stoffwechselkäfigen gehalten. Die Tiere wurden fünf Fütterungsvarianten zugeteilt, die dem Bedarf der Tiere für ein Leistungsniveau von 700 g Tageszunahmen entsprachen, in den Rationen 2 bis 5 jedoch eine P-Gehalt unter dem Tagesbedarf der Tiere aufwiesen und in den Rationen 3 bis 5 mit abgestuften Gehalten von 50, 100 sowie 200 mg/kg einer experimentellen Phytase ergänz waren. Die Absenkung des P Gehaltes im Futter verringerte den Asche- (P=0•024) und Trockenmassegehalt im Kot (P=0•017) sowie die P Konzentration im MP (P=0•000) signifikant. Die mikrobielle Biomasse im Kot wurde durch die Wiegung des MP auf durchschnittlich 467 g/kg TM bestimmt. Der Stickstoffgehalt im Kot betrug im Mittel 46•1 g/kg TM und der in die Bakterienmasse eingebaute Stickstoffanteil 27•1 g/kg TM bzw. 58% vom Gesamtstickstoffgehalt im Kot. Die BEDN Fraktion wurde auf 73% am Kotstickstoff bestimmt. Der P-Gehalt im Kot sowie der N Gehalt im MP mit durchschnittlichen 10•4 und 57•9 g/kg TM lagen im Bereich von Literaturangaben. Die P Gehalte im MP schwankten in Abhängigkeit von der Zugabe von Phytase signifikant (P=0•000) von 1•8 bis 4•8 g/kg TM. Die Aminozucker wiesen keine signifikanten unterschiede zwischen Fütterungsvarianten auf und lagen im Bereich von Werten von Rinderkot. Ergebnisse weisen darauf hin, dass die angewandte Methode zur direkten Quantifizierung der mikrobiellen Biomasse geeignet ist.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Type III secretion homologs are present in Brucella melitensis, B-ovis, and B-suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Endothelin signalling in the cardiac myocyte and its pathophysiological relevance

Endothelin A (ET(A)) transmembrane receptors predominate in rat cardiac myocytes. These are G protein-coupled receptors whose actions are mediated by the G(q) heterotrimeric G proteins. Through these, ET-1 binding to ET(A)-receptors stimulates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol remains in the membrane whereas inositol 1,4,5-trisphosphate is soluble (though its importance in the cardiac myocyte is still debated). Isoforms of the phospholipid-dependent protein kinase, protein kinase C (PKC), are intracellular receptors for diacylglycerol. Cytoplasmic nPKCdelta and nPKCepsilon detect increases in membrane diacylglycerols and translocate to the membrane. This brings about PKC activation, though modifications additional to binding to phospholipids and diacylglycerol are involved. The next event (probably associated with PKC activation) is the activation of the membrane-bound small G protein Ras by exchange of GTP for GDP. Ras.GTP loading translocates Raf family mitogen-activated protein kinase (MAPK) kinase kinases to the membrane, initiates the activation of Raf, and thus activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over longer times, two analogous protein kinase cascades, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase cascades, become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signalling molecules become phosphorylated, thus changing their biological activities. For example, ET-1 increases the expression of the c-jun transcription factor gene, and increases abundance and phosphorylation of c-Jun protein. These changes in c-Jun expression and phosphorylation are likely to be important in the regulation of gene transcription.

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

Aims Glycosylation with beta-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. Methods and results Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 mu M) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. Conclusion We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

Effect of Cluster Multi-Diode Light Emitting Diode Therapy (LEDT) on Exercise-Induced Skeletal Muscle Fatigue and Skeletal Muscle Recovery in Humans

Background and Objectives: There are some indications that low-level laser therapy (LLLT) may delay the development of skeletal muscle fatigue during high-intensity exercise. There have also been claims that LED cluster probes may be effective for this application however there are differences between LED and laser sources like spot size, spectral width, power output, etc. In this study we wanted to test if light emitting diode therapy (LEDT) can alter muscle performance, fatigue development and biochemical markers for skeletal muscle recovery in an experimental model of biceps humeri muscle contractions. Study Design/Materials and Methods: Ten male professional volleyball players (23.6 [SD +/- 5.6] years old) entered a randomized double-blinded placebo-controlled crossover trial. Active cluster LEDT (69 LEDs with wavelengths 660/850 nm, 10/30 mW, 30 seconds total irradiation time, 41.7J of total energy irradiated) or an identical placebo LEDT was delivered under double-blinded conditions to the middle of biceps humeri muscle immediately before exercise. All subjects performed voluntary biceps humeri contractions with a workload of 75% of their maximal voluntary contraction force (MVC) until exhaustion. Results: Active LEDT increased the number of biceps humeri contractions by 12.9% (38.60 [SD +/- 9.03] vs. 34.20 [SD +/- 8.68], P = 0.021) and extended the elapsed time to perform contractions by 11.6% (P = 0.036) versus placebo. In addition, post-exercise levels of biochemical markers decreased significantly with active LEDT: Blood Lactate (P = 0.042), Creatine Kinase (P = 0.035), and C-Reative Protein levels (P = 0.030), when compared to placebo LEDT. Conclusion: We conclude that this particular procedure and dose of LEDT immediately before exhaustive biceps humeri contractions, causes a slight delay in the development of skeletal muscle fatigue, decreases post-exercise blood lactate levels and inhibits the release of Creatine Kinase and C-Reative Protein. Lasers Surg. Med. 41:572-577, 2009. (C) 2009 Wiley-Liss, Inc.

Effect of periodontal treatment on metabolic control, systemic inflammation and cytokines in patients with type 2 diabetes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Nigriventrine: A low molecular mass neuroactive compound from the venom of the spider Phoneutria nigriventer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polybioside, a Neuroactive Compound from the Venom of the Social Wasp Polybia paulista

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

Insuficiência aórtica subcrônica e vias centrais relacionadas à regulação da ingestão de sódio

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biologia estrutural: expressão e caracterização estrutural da proteína 25K do Cole latent virus

Pós-graduação em Microbiologia - IBILCE

Vasoconstrictor effect of Africanized honeybee (Apis mellifera L.) venom on rat aorta

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)