Anaerobic fungal communities differ along the horse digestive tract

Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation.     

Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release

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Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes

Phospholipase D from Streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce choline and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphosphatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD toward these monolayers is tested by infrared reflection-absorption spectroscopy and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20 mN/m. On addition of C(16)OH, this strong decrease starts at 5 mN/m. For the DPPC/DPG system, the reaction yield linearly decreases between 5 and 25 mN/m. The difference in scPLD activity is correlated to the phase state of the monolayers as examined by x-ray diffraction, Brewster angle microscopy, and atomic force microscopy. Because the additives C(16)OH and DPG mediate the miscibility of PC and PA, only a basal activity of scPLD is observed toward the mixed systems at higher surface pressures. At pure DPPC monolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation. This phenomenon offers a self-regulating mechanism for the concentration of the second messenger PA within biological membranes.     

Modifying dipalmitoylphosphatidylcholine monolayers by n-hexadecanol and dipalmitoylglycerol

The monolayer structure of pure dipalmitoylphosphatidylcholine (DPPC) and equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG) are studied by the film balance technique and grazing incidence X-ray diffraction measurements. At 20 degrees C, the binary systems exhibit complete miscibility. In contrast to pure DPPC monolayers, a condensing effect is observed in the presence of both non-phospholipid additives; but the phase transition behavior differs. The tilt angle of the hydrocarbon chains in the DPPC/C(16)OH mixture is significantly smaller than in pure DPPC monolayers. The tilt of the chains is even further reduced in the mixed monolayer of DPPC/DPG. A comparison of the three systems reveals distinct structural features such as phase state, chain tilt, and molecular area over a wide range of surface pressures. Therefore, these monolayers provide a highly suitable model to investigate the influence of structural parameters on biological processes occurring at the membrane surface, e.g. enzymatic reactions and adsorption events.     

Binding of netrin-4 to laminin short arms regulates basement membrane assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.     

TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlorpromazine

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurons, where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear, but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer, causing a curvature of the membrane, which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study, we investigated the effect of these molecules on TRPA1 gating. TRPA1 was robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Regulation of the muscle-specific AMP-activated protein kinase alpha2beta2gamma3 complexes by AMP and implications of the mutations in the gamma3-subunit for the AMP dependence of the enzyme

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases.