A reliable method for detecting the intracellular accumulation of fermentation products: Application to intracellular ethanol analysis

Summary Intracellular ethanol concentrations inSaccharomyces cerevisiae were measured using a high cell density fermentation technique which permits samples to be fixed for analysis in less than two seconds. No accumulation of intracellular ethanol was detected, regardless of the stage of fermentation. The technique eliminates artifacts associated with concentrating the cell sample and provides a reliable means for detecting whether other fermentation products accumulate intracellularly.     

One-Pot Multi-Component Synthesis and Biological Evaluation of Novel Indole-Pyrimidine Derivatives as Potent Anti-Cancer and Anti-Microbial Agents

Russian Journal of Bioorganic Chemistry - A series of novel hybrids of indole-pyrimidine moieties were synthesized and evaluated for their in vitro anti-cancer, in vitro anti-bacteria and...     

Analysis of Modified Deoxynucleosides by Electrospray Ionization Mass SPECTROMETRY

Abstract

Electrospray mass spectra for selected modified deoxynucleosides and deoxynucleoside monophosphates have been determined. Protonated molecular ions are abundant in the positive ion spectrum, while (M-H) appears in the negative ion spectrum. However, fragment ion intensities are usually low in both spectra. Conditions which promote collision-induced dissociation within the electrospray source facilitate fragment ion formation, and the intensity of BH₂ ⁺ and S⁺ (positive ion spectrum) and (M-BH) and B (negative ion spectrum) are enhanced by increasing the skimmer cone voltage. MH⁺ was detected with as little as 3 pmol of deoxynucleoside, and the protonated molecular ion intensity is linear with respect to analyte concentration over two orders of magnitude.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Multiple stressor effects on coral reef ecosystems

Global climate change has profound implications on species distributions and ecosystem functioning. In the coastal zone, ecological responses may be driven by various biogeochemical and physical environmental factors. Synergistic interactions can occur when the combined effects of stressors exceed their individual effects. The Red Sea, characterized by strong gradients in temperature, salinity, and nutrients along the latitudinal axis provides a unique opportunity to study ecological responses over a range of these environmental variables. Using multiple linear regression models integrating in situ, satellite and oceanographic data, we investigated the response of coral reef taxa to local stressors and recent climate variability. Taxa and functional groups responded to a combination of climate (temperature, salinity, air‐sea heat fluxes, irradiance, wind speed), fishing pressure and biogeochemical (chlorophyll a and nutrients ‐ phosphate, nitrate, nitrite) factors. The regression model for each species showed interactive effects of climate, fishing pressure and nutrient variables. The nature of the effects (antagonistic or synergistic) was dependent on the species and stressor pair. Variables consistently associated with the highest number of synergistic interactions included heat flux terms, temperature, and wind speed followed by fishing pressure. Hard corals and coralline algae abundance were sensitive to changing environmental conditions where synergistic interactions decreased their percentage cover. These synergistic interactions suggest that the negative effects of fishing pressure and eutrophication may exacerbate the impact of climate change on corals. A high number of interactions were also recorded for algae, however for this group, synergistic interactions increased algal abundance. This study is unique in applying regression analysis to multiple environmental variables simultaneously to understand stressor interactions in the field. The observed responses have important implications for understanding climate change impacts on marine ecosystems and whether managing local stressors, such as nutrient enrichment and fishing activities, may help mitigate global drivers of change.     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.     

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.     

Tomographic phase microscopy

We report a technique for quantitative three-dimensional (3D) mapping of refractive index in live cells and tissues using a phase-shifting laser interferometric microscope with variable illumination angle. We demonstrate tomographic imaging of cells and multicellular organisms, and time-dependent changes in cell structure. Our results will permit quantitative characterization of specimen-induced aberrations in high-resolution microscopy and have multiple applications in tissue light scattering.     

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.