Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae

Signal transduction pathways control cellular responses to extrinsic and intrinsic signals. The yeast HOG (High Osmolarity Glycerol) response pathway mediates cellular adaptation to hyperosmotic stress. Pathway architecture as well as the flow of signal have been studied to a very high degree of detail. Recently, the yeast HOG pathway has become a popular model to analyse systems level properties of signal transduction. Those studies addressed, using experimentation and modelling, the role of basal signalling, robustness against perturbation, as well as adaptation and feedback control. These recent findings provide exciting insight into the higher control levels of signalling through this MAPK system of potential general importance.     

The use of digital images to evaluate the interobserver agreement on cervical smear readings in Italian cervical cancer screening

G. Tinacci, A. Biggeri, A. Pellegrini, M.P. Cariaggi, M.L. Schiboni and M. Confortini The use of digital images to evaluate the interobserver agreement on cervical smear readings in Italian cervical cancer screening Objective: The aim of this study was to measure interobserver agreement among cytologists on using a set of digital images. Methods: A set of 90 selected Papanicolaou‐stained cervical smears were digitalized and the digital images circulated among 117 readers, from laboratories spread across almost all Italian regions. Three representative fields of each smear were displayed at 20× and 40× magnification (overall six images for each case). The diagnoses made by the cytologists who provided the images were taken as target diagnoses. Results: The κ values were: very low for the categories atypical squamous cells of undetermined significance (ASC‐US), and atypical squamous cells – cannot exclude high‐grade squamous intraepithelial lesion (ASC‐H); poor for the categories atypical glandular cells (AGC), high‐grade squamous intraepithelial lesion (HSIL) and invasive cancer; and fair to good for the categories negative and low‐grade squamous intraepithelial lesion (LSIL). However, we found a cluster of 42 best concordant readers. The overall κ value and overall weighted κ with the target diagnosis for these 42 readers were 0.45 and 0.66, respectively. This finding is in contrast with the overall κ value and overall weighted κ for the other readers of 0.39 and 0.59, respectively. Conclusions: As this finding is an estimate of the accuracy of the readers, we can infer that it will be very important to reach this level of concordance for all the participating readers. Future effort will facilitate common experiences in order to improve the reproducibility of diagnostic criteria. Digital images could be the key to reach this aim.     

An Unexpected Major Groove Binding of Netropsin and Distamycin A to tRNAphe

Abstract

Crystalline complexes of yeast tRNA^phe and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 0(6), U52 0(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

A microbial sensor for discovering structural probes of protein misfolding and aggregation

In all cell types, protein homeostasis, or “proteostasis,” is maintained by sophisticated quality control networks that regulate protein synthesis, folding, trafficking, aggregation, disaggregation, and degradation. In one notable example, Escherichia coli employ a proteostasis system that determines whether substrates of the twin-arginine translocation (Tat) pathway are correctly folded and thus suitable for transport across the tightly sealed cytoplasmic membrane. Herein, we review growing evidence that the Tat translocase itself discriminates folded proteins from those that are misfolded and/or aggregated, preferentially exporting only the former. Genetic suppressors that inactivate this mechanism have recently been isolated and provide direct evidence for the participation of the Tat translocase in structural proofreading of its protein substrates. We also discuss how this discriminatory “folding sensor” has been exploited for the discovery of structural probes (e.g., sequence mutations, pharmacologic chaperones, intracellular antibodies) that modulate the folding and solubility of virtually any protein-of-interest, including those associated with aggregation diseases (e.g., α-synuclein, amyloid-β protein). Taken together, these studies highlight the utility of engineered bacteria for rapidly and inexpensively uncovering potent anti-aggregation factors.     

Body temperatures of Namib Desert tenebrionid beetles: their relationship in laboratory and field

Abstract The body temperatures of six apterous species of Namib Desert tenebrionid beetles were measured continuously with indwelling thermocouples under laboratory conditions and in the field. The range of body temperatures selected was within the upper half of their 'tolerated range', which we defined as the temperatures lying between measured critical thermal maximum and critical thermal minimum. In the field, individuals also maintained their body temperatures within the upper half of the 'tolerated range'. These beetles maintained higher body temperatures than those recorded for any other ectothermic insect. Three of the six species maintained lower body temperatures in the field than they selected in the laboratory. The other three species showed no significant difference between field and laboratory body temperatures. We conclude that these beetles are not forced by biotic or abiotic factors to adopt thermal niches which present them with physiological difficulties.     

Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

Screening of Drugs That Suppress Ste11 MAPKKK Activation in Yeast Identified a c-Abl Tyrosine Kinase Inhibitor

The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both α-factor and Ste4 G_β overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.