A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells

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Altered -3 substrate specificity of Escherichia coli signal peptidase 1 mutants as revealed by screening a combinatorial peptide library

Signal peptidase functions to cleave signal peptides from preproteins at the cell membrane. It has a substrate specificity for small uncharged residues at -1 (P1) and aliphatic residues at the -3 (P3) position. Previously, we have reported that certain alterations of the Ile-144 and Ile-86 residues in Escherichia coli signal peptidase I (SPase) can change the specificity such that signal peptidase is able to cleave pro-OmpA nuclease A in vitro after phenylalanine or asparagine residues at the -1 position (Karla, A., Lively, M. O., Paetzel, M. and Dalbey, R. (2005) J. Biol. Chem. 280, 6731-6741). In this study, screening of a fluorescence resonance energy transfer-based peptide library revealed that the I144A, I144C, and I144C/I86T SPase mutants have a more relaxed substrate specificity at the -3 position, in comparison to the wild-type SPase. The double mutant tolerated arginine, glutamine, and tyrosine residues at the -3 position of the substrate. The altered specificity of the I144C/I86T mutant was confirmed by in vivo processing of pre-beta-lactamase containing non-canonical arginine and glutamine residues at the -3 position. This work establishes Ile-144 and Ile-86 as key P3 substrate specificity determinants for signal peptidase I and demonstrates the power of the fluorescence resonance energy transfer-based peptide library approach in defining the substrate specificity of proteases.     

Genome-wide analysis of nucleotide-level variation in commonly used Saccharomyces cerevisiae strains

Ten years have passed since the genome of Saccharomyces cerevisiae-more precisely, the S288c strain-was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, summation 1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the approximately 12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers.     

Determination of roxithromycin in human plasma by HPLC with fluorescence and UV absorbance detection: application to a pharmacokinetic study

A selective HPLC method with fluorescence detection for the determination of roxithromycin (ROX) in human plasma was described. After solid-phase extraction (SPE), ROX and erythromycin (internal standard, I.S.) were derivatized by treatment with 9-fluorenylmethyl chloroformate (FMOC-Cl). Optimal resolution of fluorescence derivatives of ROX and I.S. was obtained during one analytical run using reversed phase, C(18) column. The mobile phase was composed of potassium dihydrogenphosphate solution, pH 7.5 and acetonitrile. Fluorescence of the compounds was measured at the maximum excitation, 255 nm and emission, 313 nm, of ROX derivatives. Validation parameters of the method were also established. After SPE, differences in recoveries of ROX and erythromycin from human plasma were observed. The linear range of the standard curve of ROX in plasma was 0.5-10.0 mg/l. The validated method was successfully applied for pharmacokinetic studies of ROX after administration of a single tablet of ROX.     

Determination of treosulfan in plasma and urine by HPLC with refractometric detection; pharmacokinetic studies in children undergoing myeloablative treatment prior to haematopoietic stem cell transplantation

A direct and selective HPLC method with refractometric detection was worked out for determination of treosulfan in plasma and urine of children. Before injection onto reverse phase column plasma samples with treosulfan and barbital (I.S.) were clarified using filtration. The mobile phase was composed of phosphate buffer, pH 5 and acetonitrile. The linear range of the standard curve of treosulfan spanned concentrations of 10.0-2000.0 microg/ml and 50.0-10000.0 microg/ml in plasma and urine, respectively, and covered the levels found in biological fluids after infusion of the drug. The limit of detection amounted to 5 microg/ml for plasma and 25 microg/ml for urine. Intra- and inter-day precision and accuracy of the measurement fulfilled analytical criteria accepted in pharmacokinetic studies. Recovery of treosulfan as well as stability in biological fluids was also calculated. The validated method was successfully applied in pharmacokinetic studies of treosulfan administered to children prior to haematopoietic stem cell transplantation. Differences between pharmacokinetics of treosulfan in children and adults were also studied.     

Microfungal community structure from forest soils in Northern Thrace Region, Turkey

The soil fungi in the pure stand of oak (Quercus petraea), beech (Fagus orientalis), and pine (Pinus nigra) were investigated by the dilution plate method at Yildiz Mountain in Thrace region. The mycobiota, as well as the number of isolates per plate, was determined at various soil depths. Principal component analysis of the soil profiles indicated that there was variation in mycobiota composition and the variation was attributed to differences among the ecosystems. When comparing conifer and hardwood soils, using Sorenson’s similarity index, fungal community composition corresponded more closely between the hardwood stands than with the conifer stand. Fungal community composition appears to be influenced by the organic compounds entering soil from plant litter.     

Perinatal physiology in cloned and normal calves: hematologic and biochemical profiles

Although a majority of clones are born normal and apparently healthy, mortality rates of nearly 30% are described in many reports. Such losses are a major limitation of cloning technology and represent substantial economic investment as well as justifiable animal health and welfare concerns. Prospective, controlled studies are needed to understand fully the causes of neonatal mortality in clones and to develop preventive and therapeutic strategies to minimize losses. We report here the findings of studies on the hematologic and biochemical profiles of cloned and control calves in the immediate 48-h postpartum period. Cloned calves were similar to control calves for a majority of parameters studied including blood gases, concentrations of plasma proteins, minerals and electrolytes, and white blood cell, neutrophil, lymphocyte, and platelet counts. The most notable differences between clones and controls in this study were reduced red- and white-blood cell counts in clones at birth and 1 h of age. As a group, plasma electrolyte concentrations were more variable in clones, and the variability tended to be shifted either higher (sodium, chloride) or lower (potassium, bicarbonate) than in controls. Previously, we noted differences in carbohydrate parameters, the length of time required for clones to make the neonatal adaptation to life ex utero, and morphology of the cloned placenta. Taken together, our findings suggest that cloned calves experience greater difficulty adjusting to life ex utero and that further research is warranted to determine the nature of the relationship between the physiological differences noted here in clones at birth and concomitant abnormal placental morphology.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.