Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 ?-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.     

Zur Blütenmorphologie und Bestäubungsökologie vonVeratrum album subsp.lobelianum (Bernh.)Rchb.

Zusammenfassung BeiVeratrum album subsp.lobelianum (Bernh.)Rchb. wurden im Gebirge Krkonoe (Riesengebirge) Blütenbau, Blütedauer und Blühverlauf studiert; besondere Berücksichtigung fanden dabei Vorkommen von Zwitterblüten und eingeschlechtlichen Blüten, Bau des Nektariums, Art und Weise der Nektarsekretion, Funktionsdauer der Narbe und Absinken der Keimfähigkeit des Pollens während der Anthese.Die Blütedauer der Zwitterblüten läßt eine deutliche Abhängigkeit von der Höhe ü. d. M. erkennen.Die (im Gegensatz zuVeratrum nigrum L.) weniger ausgeprägte Dichogamie erlaubt nur, ein männliches und ein Zwitterstadium der Blüte zu unterscheiden.Künstliche Bestäubungsversuche zeigten, daßV. album subsp.lobelianum weitgehend selbstfertil ist.Als regelmäßige Blumenbesucher und hauptsächlichste Bestäuber vonV. album susp.lobelianum wurden im obgenannten Gebirge 15 Dipteren-Arten (der Besucherzahl nach vorherrschend Aasfliegen) festgestellt, die ausgiebig Nachbar- und Fremdbestäubung (Geitonogamie, Allogamie), weniger schon Selbstbestäubung (Autogamie) bewirken.Die experimentelle Analyse des Blumenbesuches dieser Fliegen-Arten im Freiland ergab, daß ihre Fernanlockung durch den spezifischen Duft erfolgt, wobei die optische Wirkung der Blüten (Farbe, Form, Glanz der Nektarschicht) entbehrlich erscheint. Veratrum album susp.lobelianum kann bis zu einem gewissen Grade als aasblumig und ihre Blüte als Täuschblume bezeichnet werden.     

Seed banks on Attalea phalerata (Arecaceae) stems in the Pantanal wetland, Brazil

Background and Aims

Seeds can accumulate in the soil or elsewhere, such as on the stems of palms when these are covered by persistent sheaths. These sheaths could act as a safe site for some species. Here, we studied whether persistent sheaths of the palm Attalea phalerata (Arecaceae) are available sites for seed accumulation in the Pantanal wetland of Brazil. We also investigated whether the composition, richness and diversity of species of seeds in the persistent sheaths are determined by habitat (riparian forest and forest patches) and/or season (wet and dry).

Methods

All accumulated material was collected from ten persistent sheaths along the stems of 64 A. phalerata individuals (16 per habitat and 16 per season). The material was then individually inspected under a stereomicroscope to record seed species and number.

Key Results

Of the 640 sheaths sampled, 65 % contained seeds (n = 3468). This seed bank included 75 species belonging to 12 families, and was primarily composed of small, endozoochoric seeds, with a few abundant species (Cecropia pachystachya and Ficus pertusa). Moraceae was the richest family (four species) and Urticaceae the most abundant (1594 seeds). Stems of A. phalerata in the riparian forest had 1·8 times more seeds and 1·3 times more species than those in forest patches. In the wet season we sampled 4·1 times more seeds and 2·2 more species on palm stems than in the dry season. Richness did not differ between habitats, but was higher in the wet season. Abundance was higher in forest patches and in the wet season.

Conclusions

Attalea phalerata stems contain a rich seed bank, comparable to soil seed banks of tropical forests. As most of these seeds are not adapted to grow in flooding conditions, palm stems might be regarded as safe sites for seeds (and seedlings) to escape from the seasonal flooding of the Pantanal.     

Chlorophyll fluorescence quenching in the alga Euglena gracilis

When far red light preincubated cells of Euglena gracilis are transferred to dark or light, chlorophyll fluorescence (F₀ and F_m) decreases. Non-photochemical quenching in the dark is suggested to be induced partly by chlororespiration and partly by changes in the distribution of excitation energy between the photosystems. Depending on the light intensities it was possible to resolve the non-photochemical quenching into at least three different components. The slowest relaxation phase of non-photochemical quenching occurred only after exposure to high light and was assigned to photoinhibition. The other two components were an energy-dependent quenching (qE), and the one which we attribute to a spill over mechanism. We suggest that both photosystems use a common antenna system consisting of LHC I and LHC II proteins. In contrast to higher plants, qE in Euglena gracilis is independent of the xanthophyll cycle and an aggregation of LHC II. This revised version was published online in June 2006 with corrections to the Cover Date.     

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ～70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.     

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg²⁺ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NR_act) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NR_max). Both values together permit an estimation of the NR-phosphorylation state. Changes in NR_act and NR_max were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NR_max decreased slowly without a large change in the phosphorylation state. This decrease of NR_max was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NR_max without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NR_max and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation. Received: 29 August 1996 / Accepted: 8 November 1996     

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Structural determinants of substrate access to the disulfide oxidase Erv2p

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.     

Effects of periodic desiccation on the synthesis of the UV-screening compound, scytonemin, in cyanobacteria

Scytonemin is an ultraviolet radiation (UVR)-screening compound synthesized by some sheathed cyanobacteria exposed to high solar and sky radiation. It is primarily produced in response to UVA radiation, but certain environmental stresses can enhance synthesis. This study focuses on the effects of periodic desiccation on scytonemin synthesis in three desiccation-tolerant cyanobacterial strains, Nostoc punctiforme PCC 73102, Chroococcidiopsis CCMEE 5056 and Chroococcidiopsis CCMEE 246. Nostoc punctiforme and Chroococcidiopsis CCMEE 5056 exposed to UVA radiation produced more concentrated scytonemin screens when experiencing periodic desiccation (i.e. 1 day desiccated for every 2 days hydrated) than when continuously hydrated. A more concentrated scytonemin screen would reduce the amount of UVR damage accrued when cells are desiccated and metabolically inactive. This might allow the cyanobacteria to allocate more energy to systems other than UVR damage repair during rehydration, which would facilitate recovery. The scytonemin screen is extremely stable, remaining largely intact in the sheaths of desiccated N. punctiforme even when continuously exposed to UVA radiation for about 2 months. In contrast to the above findings, scytonemin synthesis in Chroococcidiopsis CCMEE 246, a strain that produces scytonemin constitutively under low visible light (no UVA), was partially inhibited by periodic desiccation.