Method for Obtaining Antifungal and Herbicidal Compounds that Target the First Committed Step in Shingolipid Long-Chain Base Biosynthesis

The invention provides the LCB1 and LCB2 genes of the yeast Saccharomyces cerevisiae that encode subunits of the enzyme serine palmitoyltransferase (SPT), the first enzyme leading to synthesis of the long-base component of the sphingolipids. The present specification describes the isolation of the LCB1 and LCB2 genes. The invention further relates to methods of using these genes to either inhibit SPT activity or to inhibit synthesis of the enzyme. Furthermore, the invention relates to methods for constructing strains of S. cerevisiae or other organisms that can be used to select and to test for compounds that either inhibit SPT activity or to inhibit synthesis of the enzyme

Fungal IPC Synthase Assay

The presently-disclosed IPC synthase-inhibitor assays comprise the steps of: (1) expression of the IPC1 gene in a cell; (2) introducing labeled starting substrates for ceramide conversion as well as potential inhibitor(s) of such conversion to the expressed gene product in an environment which allows time and conditions for conversion, and (3) identifying those potential inhibitors which actually inhibit conversion. The present invention also provides methods to determine the ability of a test compound to inhibit fungal growth, comprising the steps of (1) presenting active inositolphosophotidylceramide synthase in a manner such that synthesis of inositolphosphotidylceramide can occur; (2) introducing ceramide and phosphotylinositol, said ceramide or phosphatidylinositol carrying label for identification; (3) subjecting said active inositolphosophotidylceramide synthase, ceramide and phosphotylinositol to ordinary conditions necessary for ceramide conversion to phosphoinositol ceramide; and (4) identifying those test compounds which inhibit ceramide conversion to phosphoinositol ceramide

RFLP markers and genetic linkage of oil content and hypodermis color in sunflower seed (Helianthus annuus L.)

The service sector is dependent upon customers’ willingness to contribute their knowledge, skills, and abilities to co-produce the service experiences they want and expect. Service organizations therefore seek to employ strategies that will enhance their customers’ ability to do whatever they must to be successful in co-producing those experiences. Applying the concept of self-efficacy, we offer a theory-based approach to developing these strategies that firms may utilize. These strategies involve focusing both employee training and environmental cues on how to enhance the self-efficacy of the customer in performing whatever tasks are necessary toward a successful service experience

LAC+ Saccharomyces Cerevisiae

The invention relates to novel, transformed strains of Lac+ Saccharomyces cerevisiae, capable of utilizing lactose as a sole carbon source, produced by inserting into the Saccharomyces cerevisiae a plasmid containing a lactose permease and a beta-galactosidase gene derived from Kluyveromyces lactis yeast

Signal transduction protocols

Making Protein Immunoprecipitates Elaine A. Elion and Yunmei Wang Signal Transduction Inhibitors in Cellular Function Maofu Fu, Chenguang Wang, Xueping Zhang, and Richard G. Pestell Two-Dimensional Gel Electrophoresis for the Identification of Signaling Targets Yukihito Kabuyama, Kirsi K. Polvinen, Katheryn A. Resing, and Natalie G. Ahn A High-Throughput Mammalian Cell-Based Transient Transfection Assay Daniel J. Noonan, Kenneth Henry, and Michelle L. Twaroski Determining Protein Half-Lives Pengbo Zhou Assaying Protein Kinase Activity Jan Brabek and Steven K. Hanks Comparative Phosphorylation Site Mapping From Gel-Derived Proteins Using a Multidimensional ES/MS-Based Approach Francesca Zappacosta, Michael J. Huddleston, and Roland S. Annan Studies of Calmodulin-Dependent Regulation Paul C. Brandt and Thomas C. Vanaman Measurement of Protein-DNA Interactions In Vivo by Chromatin Immunoprecipitation Hogune Im, Jeffrey A. Grass, Kirby D. Johnson, Meghan E. Boyer, Jing Wu, and Emery H. Bresnick Characterization of Protein-DNA Association In Vivo by Chromatin Immunoprecipitation Laurent Kuras Nonradioactive Methods for Detecting Activation of Ras-Related Small G Proteins Douglas A. Andres Nucleocytoplasmic Glycosylation, O-GlcNAc: Identification and Site Mapping Natasha Elizabeth Zachara, Win Den Cheung, and Gerald Warren Hart Techniques in Protein Methylation Jaeho Lee, Donghang Cheng, and Mark T. Bedford Assaying Lipid Phosphate Phosphatase Activities Gil-Soo Han and George M. Carman Assaying Phosphoinositide Phosphatases Gregory S. Taylor and Jack E. Dixon Assaying Phospholipase A2 Activity Christina C. Leslie and Michael H. Gelb Measurement and Immunofluorescence of Cellular Phosphoinositides Hiroko Hama, Javad Torabinejad, Glenn D. Prestwich, and Daryll B. DeWald Measuring Dynamic Changes in cAMP Using Fluorescence Resonance Energy Transfer Sandrine Evellin, Marco Mongillo, Anna Terrin, Valentina Lissandron, and Manuela Zaccolo In Vivo Detection of Protein-Protein Interaction in Plant Cells Using BRET Chitra Subramanian, Yao Xu, Carl Hirschie Johnson, and Albrecht G. von Arnim Revealing Protein Dynamics by Photobleaching Techniques Frank van Drogen and Matthias Peter Assaying Cytochrome c Translocation During Apoptosis Nigel J. Waterhouse, Rohan Steel, Ruth Kluck, and Joseph A. Trapani Inde

Technique for Specifying the Fatty Acid at the SN2 Position of Acylglycerol Lipids

A method for specifying a fatty acid at the sn2 position of acylglycerol lipids including (a) transfecting a vector including the SLC1 gene or a variant thereof into embryonic biological material, and (b) allowing the SLC1 gene to specify the type of fatty acid at the sn2 position of acylglycerol lipids. Also provided for is an isolated SLC1 gene and a probe for its detection

Down-Regulating Sphingolipid Synthesis Increases Yeast Lifespan

Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate on ways that sphingolipids might mediate these aspects of increased lifespan. Sch9 and its mammalian homolog S6 kinase work downstream of the target of rapamycin, TOR1, protein kinase, and play evolutionarily conserved roles in regulating lifespan. Our data establish Sch9 as a focal point for regulating lifespan by integrating nutrient signals from TOR1 with growth and stress signals from sphingolipids. Sphingolipids are found in all eukaryotes and our results suggest that pharmacological down-regulation of one or more sphingolipids may provide a means to reduce age-related diseases and increase lifespan in other eukaryotes

Purification and Characterization of meta-Cresol Purple for Spectrophotometric Seawater pH Measurements

Spectrophotometric procedures allow rapid and precise measurements of the pH of natural waters. However, impurities in the acid–base indicators used in these analyses can significantly affect measurement accuracy. This work describes HPLC procedures for purifying one such indicator, meta-cresol purple (mCP), and reports mCP physical–chemical characteristics (thermodynamic equilibrium constants and visible-light absorbances) over a range of temperature (T) and salinity (S). Using pure mCP, seawater pH on the total hydrogen ion concentration scale (pHT) can be expressed in terms of measured mCP absorbance ratios (R = λ2A/λ1A) as follows:where −log(K2Te2) = a + (b/T) + c ln T – dT; a = −246.64209 + 0.315971S + 2.8855 × 10–4S2; b = 7229.23864 – 7.098137S – 0.057034S2; c = 44.493382 – 0.052711S; d = 0.0781344; and mCP molar absorbance ratios (ei) are expressed as e1 = −0.007762 + 4.5174 × 10–5T and e3/e2 = −0.020813 + 2.60262 × 10–4T + 1.0436 × 10–4 (S – 35). The mCP absorbances, λ1A and λ2A, used to calculate R are measured at wavelengths (λ) of 434 and 578 nm. This characterization is appropriate for 278.15 ≤ T ≤ 308.15 and 20 ≤ S ≤ 40

Cell-associated bacteria in the human lung microbiome

Abstract Background Recent studies have revealed that bronchoalveolar lavage (BAL) fluid contains previously unappreciated communities of bacteria. In vitro and in vivo studies have shown that host inflammatory signals prompt bacteria to disperse from cell-associated biofilms and adopt a virulent free-living phenotype. The proportion of the lung microbiota that is cell-associated is unknown. Results Forty-six BAL specimens were obtained from lung transplant recipients and divided into two aliquots: ‘whole BAL’ and ‘acellular BAL,’ the latter processed with a low-speed, short-duration centrifugation step. Both aliquots were analyzed via bacterial 16S rRNA gene pyrosequencing. The BAL specimens represented a wide spectrum of lung health, ranging from healthy and asymptomatic to acutely infected. Bacterial signal was detected in 52% of acellular BAL aliquots, fewer than were detected in whole BAL (96%, p ≤ 0.0001). Detection of bacteria in acellular BAL was associated with indices of acute infection [BAL neutrophilia, high total bacterial (16S) DNA, low community diversity, p < 0.01 for all] and, independently, with low relative abundance of specific taxonomic groups (p < 0.05). When whole and acellular aliquots from the same bronchoscopy were directly compared, acellular BAL contained fewer bacterial species (p < 0.05); whole and acellular BAL similarity was positively associated with evidence of infection and negatively associated with relative abundance of several prominent taxa (p < 0.001). Acellular BAL contained decreased relative abundance of Prevotella spp. (p < 0.05) and Pseudomonas fluorescens (p < 0.05). Conclusions We present a novel methodological and analytical approach to the localization of lung microbiota and show that prominent members of the lung microbiome are cell-associated, potentially via biofilms, cell adhesion, or intracellularity.http://deepblue.lib.umich.edu/bitstream/2027.42/111056/1/40168_2014_Article_75.pd