Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.     

IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase

Signalling by cGMP-dependent protein kinase type I (cGKI) relaxes various smooth muscles modulating thereby vascular tone and gastrointestinal motility. cGKI-dependent relaxation is possibly mediated by phosphorylation of the inositol 1,4,5-trisphosphate receptor I (IP(3)RI)-associated protein (IRAG), which decreases hormone-induced IP(3)-dependent Ca(2+) release. We show now that the targeted deletion of exon 12 of IRAG coding for the N-terminus of the coiled-coil domain disrupted in vivo the IRAG-IP(3)RI interaction and resulted in hypomorphic IRAG(Delta12/Delta12) mice. These mice had a dilated gastrointestinal tract and a disturbed gastrointestinal motility. Carbachol- and phenylephrine-contracted smooth muscle strips from colon and aorta, respectively, of IRAG(Delta12/Delta12) mice were not relaxed by cGMP, while cAMP-mediated relaxation was unperturbed. Norepinephrine-induced increases in [Ca(2+)](i) were not decreased by cGMP in aortic smooth muscle cells from IRAG(Delta12/Delta12) mice. In contrast, cGMP-induced relaxation of potassium-induced smooth muscle contraction was not abolished in IRAG(Delta12/Delta12) mice. We conclude that cGMP-dependent relaxation of hormone receptor-triggered smooth muscle contraction essentially depends on the interaction of cGKI-IRAG with IP(3)RI.     

Species specificity in avian sperm:perivitelline interaction

The interaction of chicken spermatozoa with the inner perivitelline layer from different avian species in vitro during a 5 min co-incubation was measured as the number of points of hydrolysis produced per unit area of inner perivitelline layer. The average degree of interaction, as a proportion of that between chicken spermatozoa and their homologous inner perivitelline layer, was: equal to or greater than 100% within Galliformes (chicken, turkey, quail, pheasant, peafowl and guineafowl); 44% within Anseriformes (goose, duck); and less than 30% in Passeriformes (Zebra Finch) and Columbiformes (collared-dove). The homologue of the putative chicken sperm-binding proteins, chicken ZP1 and ZP3, were identified by Western blotting with anti-chicken ZP1/ZP3 antibody in the perivitelline layers of all species. The functional cross-reactivity between chicken spermatozoa and heterologous inner perivitelline layer appeared to be linked to known phylogenetic distance between the species, although it was not related to the relative affinity of the different ZP3 homologues for anti-chicken ZP3. This work demonstrates that sperm interaction with the egg investment does not represent such a stringent species-specific barrier in birds as it does in mammals and marine invertebrates. This may be a factor in the frequency of hybrid production in birds.     

Comparison of species diversity of arbuscular mycorrhizal fungi in winter-rainfall areas of South Africa and summer-rainfall areas of Namibia

Arbuscular mycorrhizal fungi and their mycorrhizae were studied in winter rainfall areas of western South Africa. The species composition, spore numbers, and mycorrhization were attempted to correlate with various abiotic factors. Mycorrhization of grasses was significantly correlated with pH and spore numbers and negatively correlated with electric conductivity, total N-content, and total P-content of the soils. Spore numbers were negatively correlated with electric conductivity and total N-content. These relationships observed for the winter rainfall area were compared to data available for summer rainfall areas in Namibia, where the same amount of rain falls. Factors influencing species distribution were geographic distance and to a lesser extent rainfall. Rainfall was a stronger determinant of species composition in the summer rainfall area than in the winter rainfall area. These findings show that abiotic factors alone are not sufficient in explaining species distribution, and that other factors (e.g. the availability of suitable host plants) have to be taken into account when comparing different study sites.     

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.     

Mnd1 is required for meiotic interhomolog repair

BACKGROUND: While double-strand break (DSB) repair is vital to the survival of cells during both meiosis and mitosis, the preferred mechanism of repair differs drastically between the two types of cell cycle. Thus, during meiosis, it is the homologous chromosome rather than the sister chromatid that is used as a repair template. RESULTS: Cells attempting to undergo meiosis in the absence of Mnd1 arrest in prophase I due to the activation of the Mec1 DNA-damage checkpoint accumulating hyperresected DSBs and aberrant synapsis. Sporulation of mnd1Delta strains can be restored by deleting RED1 or HOP1, which permits repair of DSBs by using the sister chromatid as a repair template. Mnd1 localizes to chromatin as foci independently of DSB formation, axial element (AE) formation, and synaptonemal complex (SC) formation and does not colocalize with Rad51. Mnd1 does not preferentially associate with hotspots of recombination. CONCLUSIONS: Our results suggest that Mnd1 acts specifically to promote DSB repair by using the homologous chromosome as a repair template. The presence of Rec8, Red1, or Hop1 renders Mnd1 indispensable for DNA repair, presumably through the establishment of interhomolog (IH) bias. Localization studies suggest that Mnd1 carries out this function without being specifically recruited to the sites of DNA repair. We propose a model in which Mnd1 facilitates chromatin accessibility, which is required to allow strand invasion in meiotic chromatin.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

Synthesis and antimicrobial activity of tetrodecamycin partial structures

An efficient synthetic approach to the core structure 5 of the novel polyketide antibiotic tetrodecamycin (1) was developed. This approach features the acid-catalyzed cyclization of a tert-butyldimethylsilyl protected methyl alpha-(gamma-hydroxyacyl) tetronate, leading to the novel tricyclic ring skeleton exhibited by 5, and an efficient strategy for the parallel introduction of the cis-diol and exo-methylene function. In addition to 5, diastereomer 26, analogue 6 and several derivatives (16, 27-29) were prepared and evaluated for their antibacterial activities against Staphylococcus aureus (including MRSA) and Enterococcus faecalis and for their cytotoxic activities against human leukemia cell lines (HL-60, Jurkat T-cells). While compound 5 did not inhibit the growth of the Gram-positive pathogens (MICs >128 microg mL(-1)), analogue 6 and 2-naphthoyl derivative 27 showed promising antibacterial activities with MICs of 4-16 microg mL(-1). Remarkably, the antibacterial activity of these compounds was paralleled by cytotoxicity (IC(50) 10-23 microM). The reactive exo-methylene moiety was shown to be crucial, but not sufficient by its own, for both the antibacterial and the cytotoxic activities.     

Plasma distribution of apoA-IV in patients with coronary artery disease and healthy controls

Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).