Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42

In Saccharomyces cerevisiae cells, high external osmolarity leads to the activation of a p38-related mitogen-activated protein (MAP) kinase though Pbs2. Pbs2 tagged with green fluorescent protein (Pbs2-GFP) is evenly distributed in the cytoplasm but excluded from the nucleus before and after exposure to stress. Here we show that a catalytically inactive form of Pbs2 attains a highly polarised localization during osmostress. This phenomenon depends of the osmosensor Sho1 and on a functional Cdc42 GTPase. Cdc42, but not the actin cytoskeleton, influences Sho1-dependent activation of the MAP kinase. Sho1 itself accumulates at sites of polar growth, but independently of stress conditions and Cdc42. These observations allow us to define the sequence of events that occurs during propogation of osmostress signals.     

Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous results indicated that the modified protocol of the alkaline comet assay is a sensitive tool for the detection of formaldehyde-induced DNA-protein crosslinks. But results for mitomycin C and cisplatin suggested that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. We now used the comet assay to investigate in V79 cells the effect of potassium chromate (K(2)CrO(4)), another DNA-protein crosslinker, to see whether the results obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migration. A crosslinking effect of chromate could also not be detected with the alkaline comet assay after postincubation of cells in normal medium after chromate treatment to enable repair of other (migration-inducing) lesions that might mask the crosslinking effect. Exposure of slides to proteinase K further increased DNA migration of chromate-treated cells, thus indicating the presence of DNA-protein crosslinks. In contrast to the alkaline comet assay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma-irradiation of chromate-treated cells. The crosslinking effect was seen immediately at the end of the chromate treatment as well as after a 3h postincubation period. Using the "neutral" protocol in combination with proteinase K, we were able to demonstrate the presence of DNA-protein crosslinks as the probable cause for the migration-reducing effect. Further investigations will have to show whether this protocol can be recommended as a universal approach for the detection of DNA-protein crosslinks and also of DNA-DNA crosslinks with the comet assay.     

Kinetics of absorbance and anisotropy upon excited state relaxation in the reaction center core complex of a green sulfur bacterium

Properties of the excited states in reaction center core (RCC) complexes of the green sulfur bacterium Prosthecochloris aestuarii were studied by means of femtosecond time-resolved isotropic and anisotropic absorption difference spectroscopy at 275 K. Selective excitation of the different transitions of the complex resulted in the rapid establishment of a thermal equilibrium. At about 1 ps after excitation, the energy was located at the lowest energy transition, BChl a 835. Time constants varying between 0.26 and 0.46 ps were observed for the energy transfer steps leading to this equilibrium. These transfer steps were also reflected in changes in polarization. Our measurements indicate that downhill energy transfer towards excited BChl a 835 occurs via the energetically higher spectral forms BChl a 809 and BChl a 820. Low values of the anisotropy of about 0.07 were found in the ‘two-color’ measurements at 820 and 835 nm upon excitation at 800 nm, whereas the ‘one-color’ kinetics showed much higher anisotropies. Charge separation occurred with a time constant varying between 20 and 30 ps. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nonuniform Microtubular Polarity Established by CHO1/MKLP1 Motor Protein Is Necessary for Process Formation of Podocytes

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385–397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248–258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.     

Differentiation of isolated wheat zygotes into embryos and normal plants

Efficient and reproducible embryo development has been obtained from fertilized wheat (Triticum aestivum L.) egg cells isolated 3–6 h after hand-pollination of emasculated spikes. It is possible to routinely isolate viable zygotes from about 75% of the excised ovaries from cultivars of both winter and spring types. Co-culture with barley microspores which had been stimulated to sporophytic development resulted in embryonic development of the cultivated wheat zygotes. Within 23 h of pollination; the zygotes underwent their first cell division. They proceeded to develop into club-shaped embryos, most of which turned subsequently to dorsiventral differentiation. The morphological patterns of in-vitro-grown embryos were in accordance with those of normal zygotic embryos growing in planta. The formation of twin or multiple embryos originating from a single zygote was dependent on genotype and exogeneously supplied auxin. Upon transfer onto a suitable solidified medium, zygote-derived embryos usually germinated and developed into plants. After optimizing the feeder system, the nutrient medium and the concentration of 2,4-dichloro phenoxyacetic acid (2,4-D), more than 80 and 90% of the zygotes eventually developed into plants in genotypes Florida and Veery #5, respectively. All regenerated plants were morphologically normal and fertile. The in-vitro development from isolated zygotes of a higher-plant species into typically patterned zygotic embryos is shown here for the first time. Since the entire process, including early zygotic development, is now freely accessible to observation and micromanipulation, the method presented opens up new approaches in fundamental as well as applied fields of reproductive biology. Received: 4 September 1997 / Accepted: 28 November 1997     

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca²⁺ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca²⁺ and Mn²⁺ ions. We show here that addition of Mn²⁺ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca²⁺. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca²⁺-deficient media is overcome by expression of other Ca²⁺ pumps, including a SERCA-type Ca²⁺ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca²⁺ and Mn²⁺ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.     

High-throughput screening for ionic liquids dissolving (ligno-)cellulose

The recalcitrance of lignocellulosic biomass poses a major challenge for its sustainable and cost-effective utilization. Therefore, an efficient pretreatment is decisive for processes based on lignocellulose. A green and energy-efficient pretreatment could be the dissolution of lignocellulose in ionic liquids. Several ionic liquids were identified earlier which are capable to dissolve (ligno-)cellulose. However, due to their multitude and high costs, a high-throughput screening on small scale is essential for the determination of the most efficient ionic liquid. In this contribution two high-throughput systems are presented based on extinction or scattered light measurements. Quasi-continuous dissolution profiles allow a direct comparison of up to 96 ionic liquids per experiment in terms of their dissolution kinetics. The screening results indicate that among the ionic liquids tested EMIM Ac is the most efficient for dissolving cellulose. Moreover, it was observed that AMIM Cl is the most effective ionic liquid for dissolving wood chips.     

Midregional-proAtrial Natriuretic Peptide and High Sensitive Troponin T Strongly Predict Adverse Outcome in Patients Undergoing Percutaneous Repair of Mitral Valve Regurgitation

Background

It is not known whether biomarkers of hemodynamic stress, myocardial necrosis, and renal function might predict adverse outcome in patients undergoing percutaneous repair of severe mitral valve insufficiency. Thus, we aimed to assess the predictive value of various established and emerging biomarkers for major adverse cardiovascular events (MACE) in these patients.

Methods

Thirty-four patients with symptomatic severe mitral valve insufficiency with a mean STS-Score for mortality of 12.6% and a mean logistic EuroSCORE of 19.7% undergoing MitraClip therapy were prospectively included in this study. Plasma concentrations of mid regional-proatrial natriuretic peptide (MR-proANP), Cystatin C, high-sensitive C-reactive protein (hsCRP), high-sensitive troponin T (hsTnT), N-terminal B-type natriuretic peptide (NT-proBNP), galectin-3, and soluble ST-2 (interleukin 1 receptor-like 1) were measured directly before procedure. MACE was defined as cardiovascular death and hospitalization for heart failure (HF).

Results

During a median follow-up of 211 days (interquartile range 133 to 333 days), 9 patients (26.5%) experienced MACE (death: 7 patients, rehospitalization for HF: 2 patients). Thirty day MACE-rate was 5.9% (death: 2 patients, no rehospitalization for HF). Baseline concentrations of hsTnT (Median 92.6 vs 25.2 ng/L), NT-proBNP (Median 11251 vs 1974 pg/mL) and MR-proANP (Median 755.6 vs 318.3 pmol/L, all p<0.001) were clearly higher in those experiencing an event vs event-free patients, while other clinical variables including STS-Score and logistic EuroSCORE did not differ significantly. In Kaplan-Meier analyses, NT-proBNP and in particular hsTnT and MR-proANP above the median discriminated between those experiencing an event vs event-free patients. This was further corroborated by C-statistics where areas under the ROC curve for prediction of MACE using the respective median values were 0.960 for MR-proANP, 0.907 for NT-proBNP, and 0.822 for hsTnT.

Conclusions

MR-proANP and hsTnT strongly predict cardiovascular death and rehospitalization for HF in patients undergoing percutaneous repair of mitral valve insufficiency. Both markers might be useful components in new scoring systems to better predict short- and potentially long-term mortality and morbidity after MitraClip procedure.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.