TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.     

A combined approach to data mining of textual and structured data to identify cancer-related targets

Background  

We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas.     

Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (I(Ca,L)) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode's solution. Although depolarizations from holding potential (Vhp) -40 to 0 mV elicited relatively small inward I(Ca,L) in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of I(Ca,L) was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to -80 mV and test depolarizations were preceded by short prepulses to -40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 microM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.     

Selective synthesis of triangular cluster oxido-sulfidocomplexes of Mo and W: High yield preparations of [Mo3O2S2(H2O)9], [W3O2S2(H2O)9], [W2MoO2S2(H2O)9] and their derivatization

Reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ or metallic Mo under hydrothermal conditions (140 °C, 4 M HCl) gives oxido-sulfido cluster aqua complex [Mo₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (1). Similarly, [W₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (2) is obtained from [W₂O₂(μ-S)₂(H₂O)₆]²⁺ and W(CO)₆. While reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with W(CO)₆ mainly proceeds as simple reduction to give 1, [W₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ produces new mixed-metal cluster [W₂Mo(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (3) as main product. From solutions of 1 in HCl supramolecular adduct with cucurbit[6]uril (CB[6]) {[Mo₃O₂S₂(H₂O)₆Cl₃]₂CB[6]}Cl₂⋅18H₂O (4) was isolated and structurally characterized. The aqua complexes were converted into acetylacetonates [M₃O₂S₂(acac)₃(py)₃]PF₆ (M₃ = Mo₃, W₃, W₂Mo; 5a-c), which were characterized by X-ray single crystal analysis, electrospray ionization mass spectrometry and ¹H NMR spectroscopy. Crystal structure of (H₅O₂)(Me₄N)₄[W₃(μ₃-S)(μ₂-S)(μ₂-O)₂(NCS)₉] (6), obtained from 2, is also reported.     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Glutamatergic plasticity by synaptic delivery of GluR-B(long)-containing AMPA receptors

Activity-driven delivery of AMPA receptors is proposed to mediate glutamatergic synaptic plasticity, both during development and learning. In hippocampal CA1 principal neurons, such trafficking is primarily mediated by the abundant GluR-A subunit. We now report a study of GluR-B(long), a C-terminal splice variant of the GluR-B subunit. GluR-B(long) synaptic delivery is regulated by two forms of activity. Spontaneous synaptic activity-driven GluR-B(long) transport maintains one-third of the steady-state AMPA receptor-mediated responses, while GluR-B(long) delivery following the induction of LTP is responsible for approximately 50% of the resulting potentiation at the hippocampal CA3 to CA1 synapses at the time of GluR-B(long) peak expression-the second postnatal week. Trafficking of GluR-B(long)-containing receptors thus mediates a GluR-A-independent form of glutamatergic synaptic plasticity in the juvenile hippocampus.