The Dam1 complex confers microtubule plus end–tracking activity to the Ndc80 kinetochore complex

Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end–tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.     

Reproductive biology of Aechmea bracteata (Sw.) Griseb. (Bromelioideae: Bromeliaceae)

• Individuals of Aechmea bracteata show inflorescences with red scape bracts and odourless, yellow, tubular diurnal flowers, with closely arranged sexual organs, producing a large amount of fruits.
• In order to investigate the reproductive system of this species, a suite of characters was assessed: phenology, floral morphology and biology, nectar production dynamics, and fruit and seed production and germination, as a result of controlled pollination crosses. The study was conducted during two flowering seasons in wild populations in Yucatán, Mexico.
• Results suggest an annual flowering pattern with one flowering peak; flowers were diurnal, showing partial dichogamy (protandry)‐herkogamy, anthers and stigma become mature before floral aperture, which could lead to self‐pollination, nectar is produced during anthesis, varying in volume and total sugar concentration during the day; fruits and seeds were produced in all experimental crosses (cross‐pollination, obligated cross‐pollination, assisted and unassisted selfing, geitonogamy and apomixis), as well as high percentage seed germination.
• Several species of Aechmea are reportedly self‐compatible and autogamous, as suggested by results of selfing and non‐assisted selfing crosses, but these results are negated by the presence of apomixis, indicating that the species is apomictic. This is the first report of this breeding system for subgenus Aechmea and the sixth for Bromeliaceae. Polyembryony is here suggested for the first time in this genus and family based on the fact that more seeds were recorded that expected based on ovule numbers. Finally, when performing experimental crosses, estimating reproductive success based on number of seeds is a better approach than number of fruits, due to the effect of pseudogamy.

     

Trace element levels in mollusks from clean and polluted coastal marine sites in the Mediterranean, Red and North Seas

The trace element contamination levels in mollusks were evaluated for different marine coastal sites in the Mediterranean (Israeli coast), Red (Israeli coast) and North (German coast) Seas. Three bivalve species (Mactra corallina, Donax sp, and Mytilus edulis) and two gastropod species (Patella sp.and Cellana rota) were sampled at polluted and relatively clean sites, and their soft tissue analyzed for Hg, Cd, Zn, Cu, Mn and Fe concentrations. Representative samples were screened for organic contaminants [(DDE), polychlorinated biphenyls PCBs and polycyclic aromatic hydrocarbons (PAHs)] which exhibited very low concentrations at all sites. In the Red Sea, the gastropod C. rota showed low levels of Hg (below detection limit) and similar Cd concentrations at all the examined sites, while other trace elements (Cu, Zn, Mn, Fe) were slightly enriched at the northern beach stations. Along the Mediterranean coast of Israel, Hg and Zn were enriched in two bivalves (M. corallina and Donax sp.) from Haifa Bay, both species undergoing a long-term decrease in Hg based on previous studies. Significant Cd and Zn enrichment was detected in Patella sp. from the Kishon River estuary at the southern part of Haifa Bay. In general, Patella sp. and Donax sp. specimens from Haifa Bay exhibited higher levels of Cd compared to other sites along the Israeli Mediterranean coast, attributed to the enrichment of Cd in suspended particulate matter. Along the German coast (North Sea) M. edulis exhibited higher concentrations of Hg and Cd at the Elbe and Eider estuaries, but with levels below those found in polluted sites elsewhere. Received: 25 February 1999 / Received in revised form: 22 April 1999 / Accepted: 30 April 1999     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.     

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C₁₆ and C₁₈ fatty acids at pH ∼7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.The fungus Aspergillus nidulans (teleomorph Emericella nidulans) is a homothallic ascomycete that has a defined sexual and asexual developmental cycle. Therefore, it serves as a model system for the understanding of fungal development (1). Oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids were found to influence the development of the asexual conidiophores and sexual cleistothecia (2–6). Moreover, they seem to regulate the secondary metabolism of the fungus (7). These substances were collectively named psi factors and are primarily a mixture of hydroxylated oleic (18:1^Δ9Z; x:y^Δz denotes a fatty acid with x carbons and y double bonds in position z counting from the carboxyl end), linoleic (18:2^Δ9Z,12Z), and α-linolenic (18:3^Δ9Z,12Z,15Z) acids. They are termed psiβ, psiα, and psiγ, respectively. Psi factors can be further classified by the number and positioning of hydroxy groups on the fatty acid backbone: psiB (OH at C-8, e.g. (8R)-HODE),² psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE), and psiC (OH at C-8 and the δ-lactone ring) (8, 9).The psi factor (8R)-HODE was first discovered in the fungus Laetisaria arvalis (10, 11); it was later also found in Gaeumannomyces graminis (12, 13), where the first enzyme, which is responsible for production of (8R)-HPODE, 7,8-LDS, was detected (13). This heme-containing enzyme is bifunctional because it oxidizes 18:2^Δ9Z,12Z in a first reaction step to (8R)-HPODE and subsequently isomerizes this intermediate compound to (7S,8S)-DiHODE (13–15).After the genome of A. nidulans was available, Keller and co-workers (6, 16, 17) found three genes that share a high homology with the sequence of 7,8-LDS, namely ppoA, ppoB, and ppoC. They showed that the deletion of these genes had a significant effect (i) on the developmental ratio between the asexual conidiospores and sexual ascospores; (ii) on the production of psi factors; and (iii) on the production of secondary metabolites, the mycotoxins (6, 7, 16, 17). Furthermore, the encoded proteins showed remarkable sequence homology to both mammalian PGHS isoforms, enzymes that are responsible for the synthesis of prostaglandins (18). Using the NCBI conserved domain search analysis tool, it turned out that ppoA amino acid residues 210–580 contain a domain similar to mammalian heme peroxidases, whereas residues 650–1050 contain a CYPX domain, similar to P450 heme thiolate enzymes (16). However, for 7,8-LDS from G. graminis, only the mammalian heme peroxidase domain is predicted. The identity of conserved catalytic domains between Ppo enzymes and mammalian PGHS ranges from 25 to 29% for PGHS-2 and from 25 to 26% for PGHS-1 (19). PpoA and 7,8-LDS show 42% amino acid identity.Oliw and co-workers (20) observed that incubation of homogenates of mycelia of A. nidulans with 18:2^Δ9Z,12Z converted the fatty acid to (8R)-HODE and (5S,8R)-DiHODE as the major products. (8R)-HPODE, (10R)-HODE, and (10R)-HPODE were detected as minor products. Incubation of mycelia of Aspergillus fumigatus with deuterium-labeled 18:2^Δ9Z,12Z revealed that the synthesis of (8R)-HPODE is accomplished via pro-S-hydrogen abstraction at C-8 and antarafacial dioxygen insertion. (5S,8R)-DiHODE is generated via an additional pro-S-hydrogen abstraction at C-5 of the substrate (20, 21).Additional studies with fungal knock-out strains led to the hypothesis that PpoA may be responsible for the synthesis of (8R)-hydroperoxides, which are partially reduced to (8R)-hydroxides (20). It was suggested that, analogous with 7,8-LDS, (8R)-hydroperoxides are then converted to 5,8-dihydroxides by PpoA. Furthermore, it was concluded that ppoC may code for linoleate (10R)-DOX (20). Analysis of Ppo enzymes from A. nidulans in studies published so far has been performed either by using knock-out mutants to demonstrate the absence of a subset of psi factors or by using crude mycelial extracts; both experimental setups have the disadvantage of observing multiple enzymatic reactions in parallel.To characterize the biochemical properties of PpoA in more detail, we cloned and expressed recombinant PpoA in Escherichia coli. After purification of the enzyme by up to 62-fold, biochemical characterization was performed. The studies revealed mechanistic as well as structural similarities to and differences from 7,8-LDS from G. graminis. Both enzymes were found to be homotetrameric ferric heme proteins that catalyze the synthesis of (8R)-HPODE. Whereas G. graminis 7,8-LDS converts the intermediate formed to (7S,8S)-DiHODE, PpoA produces 5,8-DiHODE.Using site-directed mutagenesis, we provide evidence that there are striking differences between both enzymes regarding the catalytic reaction cycle. Thus, we found that PpoA uses different domains to catalyze the two reaction steps. We suggest that the DOX reaction, yielding 8-HPODE, takes place in the N-terminal heme peroxidase domain. The isomerization of this intermediate product to the end product, 5,8-DiHODE, is accomplished, however, independently by the C-terminal P450 heme thiolate domain in an 8-hydroperoxide isomerase reaction.In addition, we are able to provide evidence that, during the catalysis, PpoA generates a carbon-centered radical presumably at C-8, like G. graminis 7,8-LDS. Furthermore, we determined the kinetic parameters for the first reaction step.     

Increased exploratory activity and altered response to LSD in mice lacking the 5-HT(5A) receptor

In order to determine the distribution and function of the 5-HT5A serotonin receptor subtype, we generated knockout mice lacking the 5-HT5A gene. Comparative autoradiography studies of brains of wild-type (wt) and 5-HT5A knockout (5A-KO) mice revealed the existence of binding sites with high affinity for [125I]LSD that correspond to 5-HT5A receptors and that are concentrated in the olfactory bulb, neocortex, and medial habenula. When exposed to novel environments, the 5A-KO mice displayed increased exploratory activity but no change in anxiety-related behaviors. In addition, the stimulatory effect of LSD on exploratory activity was attenuated in 5A-KO mice. These results suggest that 5-HT5A receptors modulate the activity of neural circuits involved specifically in exploratory behavior and suggest that some of the psychotropic effects of LSD may be mediated by 5-HT5A receptors.     

Critical role of nucleotide-binding oligomerization domain-like receptor 3 in vascular repair

Vascular remodeling characterized by hyperproliferative neointima formation is an unfavorable repair process that is triggered by vascular damage. This process is characterized by an increased local inflammatory and proliferative response that critically involves the pro-inflammatory cytokine interleukin-1β (IL-1β). IL-1β is expressed and cytosolically retained as a procytokine that requires additional processing prior to exerting its pro-inflammatory function. Maturation and release of pro IL-1β is governed by a cytosolic protein scaffold that is known as the inflammasome.Here we show that NLRP3 (NOD-like receptor family, pryin domain containing 3), an important activating component of the inflammasome, is involved in neointima formation after vascular injury. NLRP3 deficiency itself does not affect the functional cardiovascular phenotype and does not alter peripheral differential blood counts. However, neointima development following wire injury of the carotid artery was significantly decreased in NLRP3-deficient mice as compared to wild-type controls. In all, NLRP3 plays a non-redundant role in vascular damage mediated neointima formation.Our data establish NLRP3 as a key player in the response to vascular damage, which could open new avenues to therapeutic intervention.