Inhibition of trehalose crystallization by cytoplasmic yeast components

The influence of different yeast (Saccharomyces cerevisiae) cellular fractions was studied in an attempt to gain knowledge on the feasibility of trehalose crystallization in yeast cells. Certain constituents of S. cerevisiae cells inhibited/delayed trehalose crystallization upon humidification at high relative humidities.     

Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and methyl-salicylate and their relation with calcium signalling

Ca²⁺ plays a critical role as second messenger in the signal–response coupling of plant defence responses, and methyl-jasmonate and methyl-salicylate are important components of signal transduction cascades activating plant defences. When intact axenic non-induced seedling roots of sunflower were treated with different Ca²⁺ concentrations up to 1 mM, there was no significant increase in O ₂ ^.? generation or DMAB–MBTH peroxidase (extracellular, ECPOX) activities in the apoplast, probably because these roots had enough Ca²⁺ in their exo- and endocellular reservoirs. Both activities were strongly inhibited by the RBOH–NADPH oxidase inhibitor DPI and by the Ca²⁺ surrogate antagonist La³⁺, but the voltage-dependent Ca²⁺ channel blocker verapamil was only inhibitory at concentrations higher than those active on animal L-type Ca²⁺ channels. Concentrations >5 mM EGTA (chelating Ca²⁺ in the apoplast) and Li⁺ (inhibiting PI cycle dependent endogenous Ca²⁺ fluxes) also inhibited both activities. W7, inhibitor of binding of Ca–CaM to its target protein, enhanced both activities, but the inactive analogue W5 showed a similar effect. Our data suggest that Ca²⁺ from exocellular and, to a lesser extent, from endocellular stores is involved in oxidative activities, and that RBOH–NADPH oxidase is the main system supporting them. Ca²⁺ activation of the PM cytosolic side of RBOH–NADPH oxidase is probably the key to Ca²⁺ involvement in these processes. Roots induced by MeJA or MeSA showed significant enhancement of both oxidative activities, as corresponding to the oxidative burst evoked by the two phytohormones in the root apoplast. But while ECPOX activity showed a response to the effectors similar to that described above for non-induced roots, O ₂ ^.? generation activity in the apoplast of induced roots was insensitive to EGTA, verapamil and Li⁺, the inhibitors of exogenous and endogenous Ca²⁺ fluxes; only DPI and La³⁺ were inhibitory. As exogenously added 0.1 mM Ca²⁺ also increased O ₂ ^.? generation, we propose that, in these roots, activation of RBOH–NADPH oxidase by Ca²⁺ could be regulated by Ca²⁺ sensors in the apoplast.     

Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains

RepB initiates plasmid rolling‐circle replication by binding to a triple 11‐bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full‐length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin‐binding and catalytic domains show a three‐layer α–β–α sandwich fold. The active site is positioned at one of the faces of the β‐sheet and coordinates a Mn²⁺ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four α‐helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.     

Ultrasonic velocity assay of extracellular invertase in living yeasts

Being the largest family of cell surface receptors, G-protein-coupled receptors (GPCRs) are among the most frequent targets. The functions of many GPCRs are unknown, and it is both time-consuming and expensive to determine their ligands and signaling pathways by experimental methods. It is of great practical significance to develop an automated and reliable method for classification of GPCRs. In this study, a novel method based on the concept of Chou’s pseudo amino acid composition has been developed for predicting and recognizing GPCRs. The discrete wavelet transform was used to extract feature vectors from the hydrophobicity scales of amino acid to construct pseudo amino acid (PseAA) composition for training support vector machine. The prediction accuracies by the current method among the major families of GPCRs, subfamilies of class A, and types of amine receptors were 99.72%, 97.64%, and 99.20%, respectively, showing 9.4% to 18.0% improvement over other existing methods and indicating that the proposed method is a useful automated tool in identifying GPCRs.     

MALDI Profiling of Human Lung Cancer Subtypes

Background

Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time.

Methodology/Principal Findings

In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree–based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non–small cell lung cancer histological subtypes.

Conclusions/Significance

A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer.     

Assessment of Biology Majors’ Versus Nonmajors’ Views on Evolution, Creationism, and Intelligent Design

The controversy around evolution, creationism, and intelligent design resides in a historical struggle between scientific knowledge and popular belief. Four hundred seventy-six students (biology majors n = 237, nonmajors n = 239) at a secular liberal arts private university in Northeastern United States responded to a five-question survey to assess their views about: (1) evolution, creationism, and intelligent design in the science class; (2) students’ attitudes toward evolution; (3) students’ position about the teaching of human evolution; (4) evolution in science exams; and (5) students’ willingness to discuss evolution openly. There were 60.6% of biology majors and 42% of nonmajors supported the exclusive teaching of evolution in the science class, while 45.3% of nonmajors and 32% of majors were willing to learn equally about evolution, creationism, and intelligent design (question 1); 70.5% of biology majors and 55.6% of nonmajors valued the factual explanations evolution provides about the origin of life and its place in the universe (question 2); 78% of the combined responders (majors plus nonmajors) preferred science courses where evolution is discussed comprehensively and humans are part of it (question 3); 69% of the combined responders (majors plus nonmajors) had no problem answering questions concerning evolution in science exams (question 4); 48.1% of biology majors and 26.8% of nonmajors accepted evolution and expressed it openly, but 18.2% of the former and 14.2% of the latter accepted evolution privately; 46% of nonmajors and 29.1% of biology majors were reluctant to comment on this topic (question 5). Combined open plus private acceptance of evolution within biology majors increased with seniority, from freshman (60.7%) to seniors (81%), presumably due to gradual exposure to upper-division biology courses with evolutionary content. College curricular/pedagogical reform should fortify evolution literacy at all education levels, particularly among nonbiologists.     

LapF,the second largest Pseudomonas putida protein,contributes to plant root colonization and determines biofilm architecture

We have investigated the role of LapF, one of the two largest proteins encoded in the genome of Pseudomonas putida KT2440, in bacterial colonization of solid surfaces. LapF is 6310 amino acids long, and is localized on the cell surface. The C‐terminal region of the protein is essential for its secretion, which presumably requires the ABC transporter encoded by an operon (lapHIJ) adjacent to the lapF gene. Although the initial attachment stages are not different between the wild type and a lapF mutant, microcolony formation and subsequent development of a mature biofilm is impaired in the mutant. This is consistent with the expression pattern of lapF; activation of its promoter takes place at late stages of growth and is regulated by the alternative sigma factor RpoS. A lapF mutant is also affected in individual and competitive plant root colonization. In these assays, mixed microcolonies formed by cells of both the wild‐type and the mutant strains could be observed but microcolonies of the mutant alone were not found. These data and the localization of the protein at discrete spots in areas of contact between cells in biofilms suggest that LapF determines the establishment of cell–cell interactions during sessile growth.     

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.