Differential effects of monensin and a blend of essential oils on rumen microbiota composition of transition dairy cows.

In response to oral application, monensin alters the rumen microbiota, increasing ruminal propionate production and energy availability in the animal. Data from different studies indicate that the susceptibility of rumen bacteria to monensin is mainly cell-wall dependent but tracing its activity to specific microbial groups has been challenging. Several studies have shown a similar effect for essential oils but results are inconsistent. To investigate the influence of monensin and a blend of essential oils (BEO, containing thymol, guaiacol, eugenol, vanillin, salicylaldehyde, and limonene) on the rumen microbiome, rumen liquid samples were collected orally on d 56 postpartum from cows that had either received a monensin controlled-release capsule 3 wk antepartum, a diet containing a BEO from 3 wk antepartum onward, or a control diet (n = 12). The samples were analyzed for pH, volatile fatty acid, ammonia, and lipopolysaccharide concentrations and protozoal counts. A 16S rRNA gene fingerprinting analysis (PCR-single-strand conformation polymorphism) and sequencing revealed that the BEO treatment had no effect on the rumen microbiota, whereas monensin decreased bacterial diversity. Twenty-three bacterial species-level operational taxonomic units were identified for which monensin caused a significant decrease in their relative abundance, all belonging to the phyla Bacteroidetes (uncultured BS11 gut group and BS9 gut group) and Firmicutes (Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae). Ten bacterial operational taxonomic units belonging to the phyla Actinobacteria (Coriobacteriaceae), Bacteroidetes (Prevotella), Cyanobacteria (SHA-109), and Firmicutes (Lachnospiraceae and Ruminococcaceae) increased in relative abundance due to the monensin treatment. These results confirm the hypothesis that varying effects depending on cell-wall constitution and thickness might apply for monensin sensitivity rather than a clear-cut difference between gram-negative and gram-positive bacteria. No effect of monensin on the archaea population was observed, confirming the assumption that reported inhibition of methanogenesis is most likely caused through a decrease in substrate availability, rather than by a direct effect on the methanogens. The data support the hypothesis that the observed increase in ruminal molar propionate proportions due to monensin may be caused by a decrease in abundance of non-producers and moderate producers of propionate and an increase in abundance of succinate and propionate producers.

The direct effects of male killer infection on fitness of ladybird hosts (Coleoptera: Coccinellidae).

Male killing bacteria are common in insects and are thought to persist in host populations primarily by indirect fitness benefits to infected females, whereas direct fitness effects are generally assumed to be neutral or deleterious. Here, we estimated the effect of male killer infection on direct fitness (number of eggs laid, as a measure of fecundity, together with survival) and other life-history traits (development time and body size) in seven ladybird host/male killer combinations. Effects of male killers on fecundity ranged, as expected, from costly to neutral; however, we found evidence of reduced development time and increased survival and body size in infected strains. Greater body size in Spiroplasma-infected Harmonia axyridis corresponded to greater ovariole number and therefore higher potential fecundity. To our knowledge, this is the first report of direct benefits of male killer infection after explicitly controlling for indirect fitness effects. Neutral or deleterious fitness effects of male killer infection should not therefore be automatically assumed.

Structural Characterisation Reveals Mechanism of IL-13-Neutralising Monoclonal Antibody Tralokinumab as Inhibition of Binding to IL-13Rα1 and IL-13Rα2.

Interleukin (IL)-13 is a pleiotropic T helper type 2 cytokine frequently associated with asthma and atopic dermatitis. IL-13-mediated signalling is initiated by binding to IL-13Rα1, which then recruits IL-4Rα to form a heterodimeric receptor complex. IL-13 also binds to IL-13Rα2, considered as either a decoy or a key mediator of fibrosis. IL-13-neutralising antibodies act by preventing IL-13 binding to IL-13Rα1, IL-4Rα and/or IL-13Rα2. Tralokinumab (CAT-354) is an IL-13-neutralising human IgG4 monoclonal antibody that has shown clinical benefit in patients with asthma. To decipher how tralokinumab inhibits the effects of IL-13, we determined the structure of tralokinumab Fab in complex with human IL-13 to 2 Å resolution. The structure analysis reveals that tralokinumab prevents IL-13 from binding to both IL-13Rα1 and IL-13Rα2. This is supported by biochemical ligand-receptor interaction assay data. The tralokinumab epitope is mainly composed of residues in helices D and A of IL-13. It is mostly light chain complementarity-determining regions that are driving paratope interactions; the variable light complementarity-determining region 2 plays a key role by providing residue contacts for a network of hydrogen bonds and a salt bridge in the core of binding. The key residues within the paratope contributing to binding were identified as Asp50, Asp51, Ser30 and Lys31. This study demonstrates that tralokinumab prevents the IL-13 pharmacodynamic effect by binding to IL-13 helices A and D, thus preventing IL-13 from interacting with IL-13Rα1 and IL-13Rα2.

Role of gap junctions in the responses to EDHF in rat and guinea-pig small arteries.

1. In guinea-pig internal carotid arteries with an intact endothelium, acetylcholine (10 microM) and levcromakalim (10 microM) each hyperpolarized the smooth muscle whereas a 5 mM elevation of extracellular K(+) was without effect. 2. Incubation of the carotid artery with the gap junction inhibitors carbenoxolone (100 microM) or gap 27 (500 microM) essentially abolished the hyperpolarization to acetylcholine but it was without effect on that to levcromakalim. Carbenoxolone had no effect on the acetylcholine-induced endothelial cell hyperpolarization but inhibited the smooth muscle hyperpolarization induced by the endothelial cell K(+) channel opener, 1-ethyl-2-benzimidazolinone (600 microM). 3. In rat hepatic and mesenteric arteries with endothelium, carbenoxolone (100 or 500 microM) depolarized the smooth muscle but did not modify hyperpolarizations induced by KCl or levcromakalim. In the mesenteric (but not the hepatic) artery, the acetylcholine-induced hyperpolarization was inhibited by carbenoxolone. 4. Phenylephrine (1 microM) depolarized the smooth muscle cells of intact hepatic and mesenteric arteries, an effect enhanced by carbenoxolone. Gap 27 did not have a depolarizing action. In the presence of phenylephrine, acetylcholine-induced hyperpolarization of both hepatic and mesenteric artery myocytes was partially inhibited by each of the gap junction inhibitors. 5. Collectively, the data suggest that gap junctions play some role in the EDHF (endothelium-derived hyperpolarizing factor) response in rat hepatic and mesenteric arteries. However, in the guinea-pig internal carotid artery, electrotonic propagation of endothelial cell hyperpolarizations via gap junctions may be the sole mechanism underlying the response previously attributed to EDHF.

Further investigation of endothelium-derived hyperpolarizing factor (EDHF) in rat hepatic artery: studies using 1-EBIO and ouabain.

1. The characteristics of endothelium-dependent hyperpolarization in rat hepatic artery have been further investigated in the presence of inhibitors of cyclo-oxygenase and nitric oxide synthase. 2. Using sharp micro-electrodes, the smooth muscle hyperpolarization induced by acetylcholine, KCl or 1-ethyl-2-benzimidazolinone (1-EBIO) in intact hepatic arteries was abolished by 30 micronM barium plus 500 nM ouabain. 3. In vessels without endothelium, the smooth muscle hyperpolarization induced by KCl was not reduced by 30 micronM barium alone. However, in the presence of barium the effects of KCl were partially inhibited by 100 nM ouabain and essentially abolished by 500 nM ouabain. 4. Using sharp micro-electrodes, the hyperpolarization of both the smooth muscle and the endothelium induced by 1-EBIO or by acetylcholine was unaffected by 100 nM iberiotoxin. However, in the presence of 100 nM charybdotoxin, the effects of 1-EBIO were abolished whereas those of acetylcholine were only partially reduced. The hyperpolarization induced by levcromakalim was unaffected by either charybdotoxin or iberiotoxin. 5 Under whole-cell patch-clamp recording conditions, 1-EBIO induced a voltage-insensitive, charybdotoxin-sensitive K+ current in cultured endothelial cells but was without effect on K+ currents in smooth muscle cells isolated from hepatic arteries. 6 It is concluded that the endothelium-dependent hyperpolarization of smooth muscle induced by either acetylcholine or by 1-EBIO in rat hepatic artery is initially associated with the opening of endothelial calcium-sensitive K+-channels insensitive to iberiotoxin. The resulting accumulation of K+ in the myoendothelial space activates an isoform of Na+/K+-ATPase which is sensitive to low concentrations of ouabain.

Further investigations into the endothelium-dependent hyperpolarizing effects of bradykinin and substance P in porcine coronary artery.

In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF). In the presence of 300 microM L-nitroarginine and 10 microM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 microM glibenclamide whereas those to NOR-1, substance P and bradykinin were unaffected. Hyperpolarizations produced by maximally-effective concentrations of NOR-1 and NS1619 were identical (to -65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately -80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin. Incubation of segments of intact arteries for 16 - 22 h in bicarbonate-buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 microM 17-octadecynoic acid. We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K(+) channels, activated only by bradykinin.

Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery.

1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.

Functional evidence of a role for two-pore domain potassium channels in rat mesenteric and pulmonary arteries.

1. Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pH(o)) change membrane potential (E(M)) in rat mesenteric and pulmonary arteries. 2. Changing pH(o) from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 microm) or bupivacaine (100 microm) reversed the hyperpolarisation associated with alkaline pH(o), shifting the E(M) of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 microm) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. 3. K(+) channel blockade by 4-aminopyridine (4-AP) (5 mm), tetraethylammonium (TEA) (10 mm), Ba(2+) (30 microm) and glibenclamide (10 microm) depolarised the pulmonary artery. However, shifts in E(M) with changes in pH(o) remained and were sensitive to anandamide (10 microm), bupivacaine (100 microm) or Zn(2+) (200 microm). 4. Anandamide (0.3-60 microm) or bupivacaine (0.3-300 microm) caused a concentration-dependent increase in basal tone in pulmonary arteries. 5. RT-PCR demonstrated the expression of TASK-1, TASK-2, THIK-1, TRAAK, TREK-1, TWIK-1 and TWIK-2 in mesenteric arteries and TASK-1, TASK-2, THIK-1, TREK-2 and TWIK-2 in pulmonary arteries. TASK-1, TASK-2, TREK-1 and TWIK-2 protein was demonstrated in both arteries by immunostaining. 6. These experiments provide evidence for the presence of two-pore domain K(+) channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK-1 channels is most likely to have mediated the pH-induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.

Analyzing variability in nectar amino acids: composition is less variable than concentration.

Thirty species of flowering plants were analyzed for floral nectar amino acid composition. High-performance liquid chromatography (HPLC) was used in conjunction with AccQtag derivatization to produce accurate and precise data. For any one species, the total concentration of amino acids varies greatly (average coefficient of variation 0.65), but composition is much less variable (average correlation among samples from a single species 0.85). Absolute concentration of individual amino acids is much more variable than the relative abundance (coefficients of variation 0.98 and 0.77, respectively; N = 544, t = 16.98, P < 0.001). When amino acids that occur in only small relative abundance (<1%) are removed from the analysis, the difference is even more marked (0.78 and 0.51, respectively; N = 344, t = 15.13, P < 0.001). The results highlight the need for large sample sizes when making observations concerning the absolute amounts of amino acids in nectar and for sensitive analyses of the composition, as even small changes may be biologically significant.

K+ is an endothelium-derived hyperpolarizing factor in rat arteries.

In arteries, muscarinic agonists such as acetylcholine release an unidentified, endothelium-derived hyperpolarizing factor (EDHF) which is neither prostacyclin nor nitric oxide. Here we show that EDHF-induced hyperpolarization of smooth muscle and relaxation of small resistance arteries are inhibited by ouabain plus Ba2+; ouabain is a blocker of Na+/K+ ATPase and Ba2+ blocks inwardly rectifying K+ channels. Small increases in the amount of extracellular K+ mimic these effects of EDHF in a ouabain- and Ba2+-sensitive, but endothelium-independent, manner. Acetylcholine hyperpolarizes endothelial cells and increases the K+ concentration in the myoendothelial space; these effects are abolished by charbdotoxin plus apamin. Hyperpolarization of smooth muscle by EDHF is also abolished by this toxin combination, but these toxins do not affect the hyperpolarizaiton of smooth muscle by added K+. These data show that EDHF is K+ that effluxes through charybdotoxin- and apamin-sensitive K+ channels on endothelial cells. The resulting increase in myoendothelial K+ concentration hyperpolarizes and relaxes adjacent smooth-muscle cells by activating Ba2+-sensitive K+ channels and Na+/K+ ATPase. These results show that fluctuations in K+ levels originating within the blood vessel itself are important in regulating mammalian blood pressure and flow.