149 research outputs found
Analysis of Diffusion of Ras2 in Saccharomyces cerevisiae Using Fluorescence Recovery after Photobleaching
Binding, lateral diffusion and exchange are fundamental dynamic processes
involved in protein association with cellular membranes. In this study, we
developed numerical simulations of lateral diffusion and exchange of
fluorophores in membranes with arbitrary bleach geometry and exchange of the
membrane localized fluorophore with the cytosol during Fluorescence Recovery
after Photobleaching (FRAP) experiments. The model simulations were used to
design FRAP experiments with varying bleach region sizes on plasma-membrane
localized wild type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S
with a single lipid anchor in live yeast cells to investigate diffusional
mobility and the presence of any exchange processes operating in the time scale
of our experiments. Model parameters estimated using data from FRAP experiments
with a 1 micron x 1 micron bleach region-of-interest (ROI) and a 0.5 micron x
0.5 micron bleach ROI showed that GFP-Ras2, single or dual lipid modified,
diffuses as single species with no evidence of exchange with a cytoplasmic
pool. This is the first report of Ras2 mobility in yeast plasma membrane. The
methods developed in this study are generally applicable for studying diffusion
and exchange of membrane associated fluorophores using FRAP on commercial
confocal laser scanning microscopes.Comment: Accepted for publication in Physical Biology (2010). 28 pages, 7
figures, 3 table
Identification of Protein Palmitoylation Inhibitors from a Scaffold Ranking Library
The addition of palmitoyl moieties to proteins regulates their membrane targeting, subcellular localization, and stability. Dysregulation of the enzymes which catalyzed the palmitoyl addition and/or the substrates of these enzymes have been linked to cancer, cardiovascular, and neurological disorders, implying these enzymes and substrates are valid targets for pharmaceutical intervention. However, current chemical modulators of zDHHC PAT enzymes lack specificity and affinity, underscoring the need for screening campaigns to identify new specific, high affinity modulators. This report describes a mixture based screening approach to identify inhibitors of Erf2 activity. Erf2 is the Saccharomyces cerevisiae PAT responsible for catalyzing the palmitoylation of Ras2, an ortholog of the human Ras oncogene proteins. A chemical library developed by the Torrey Pines Institute for Molecular Studies consists of more than 30 million compounds designed around 68 molecular scaffolds that are systematically arranged into positional scanning and scaffold ranking formats. We have used this approach to identify and characterize several scaffold backbones and R-groups that reduce or eliminate the activity of Erf2 in vitro. Here, we present the analysis of one of the scaffold backbones, bis-cyclic piperazine. We identified compounds that inhibited Erf2 auto-palmitoylation activity using a fluorescence-based, coupled assay in a high throughput screening (HTS) format and validated the hits utilizing an orthogonal gel-based assay. Finally, we examined the effects of the compounds on cell growth in a yeast cell-based assay. Based on our results, we have identified specific, high affinity palmitoyl transferase inhibitors that will serve as a foundation for future compound design
Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene
The isolation and characterization of MGM1 , and yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2 . Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46976/1/294_2004_Article_BF00324678.pd
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A new method for estimating growth rates of alkenone-producing haptophytes
Laboratory culture experiments were performed to establish the range of growth conditions where ¹³C labeling of di- and tri-unsaturated C₃₇ methyl ketones yields reliable growth rates for alkenone-producing algae. Results document that even at low growth rates and short time intervals, ¹³C labeling of the di-unsaturated C₃₇ alkenone provides reasonable estimates of growth rate for Isochrysis galbana, Isochrysis sp., and three strains of Emiliania huxleyi. These findings suggest that although alkenone biosynthesis almost certainly involves a complex combination of intermediate pools, those pools must turn over at a rate sufficiently fast that the labeling of the di-unsaturated C₃₇ alkenone is not greatly biased. However, bias was noted for the tri-unsaturated alkenone, suggesting that either growth rates in the field should be based on K₃₇:₂ labeling or that long incubations should be used. Specific growth rates calculated from alkenone ¹³C labeling experiments conducted in the subarctic Pacific decreased as a function of depth in the euphotic zone and were linearly correlated with photosynthetically active radiation below ∼50 µEin m⁻² s⁻¹. These observations indicate that at depths greater than ∼25 m, growth rates of the alkenone-producing algae were light-limited. Determination of the specific growth rates of the alkenone-producing algae was motivated by our present knowledge of the controls on stable carbon isotopic fractionation in marine microalgae. Development of our alkenone ¹³C labeling technique for in situ growth rate determinations allows evaluation of the effect of growth rate on carbon isotopic fractionation in natural populations of E. huxleyi and Gephyrocapsa oceanica so that laboratory-based microalgal stable isotope fractionation hypotheses may be evaluated in the field
Specific effects of KChIP3/calsenilin/DREAM, but not KChIPs 1, 2 and 4, on calcium signalling and regulated secretion in PC12 cells
The KChIPs (K+ channel-interacting proteins) are members of the NCS (neuronal calcium sensor) protein family of Ca2+-binding proteins. It is unclear to what extent the KChIPs have distinct functions although they all interact with Kv4 K+ channels. KChIP3 has also been shown to repress transcription of specific genes via binding to DRE (downstream regulatory element) motifs and all KChIPs may share this function. In the present study, we have compared the function of isoforms of the four KChIPs. KChIPs 1–4 were found to stimulate the traffic of Kv4.2 channels to the plasma membrane. KChIP3 expression in PC12 cells resulted in an increase in exocytosis evoked by activation of purinergic receptors. In contrast, KChIPs 1, 2 and 4, although expressed to the same extent, had no effect on secretion. In addition, KChIP3 but not KChIPs 1, 2 and 4 modified the ATP-induced Ca2+ signal resulting in a delay in recovery after the peak Ca2+ elevation and also specifically resulted in down-regulation of the Na+/Ca2+ exchanger NCX3, which could explain the effects on the Ca2+ signal and secretion. Regulation of NCX3 by KChIP3 has been shown to occur via its DREAM (DRE antagonist modulator) function [Gomez-Villafuertes, Torres, Barrio, Savignac, Gabellini, Rizzato, Pintado, Gutierrez-Adan, Mellstrom, Carafoli and Naranjo (2005) J. Neurosci. 25, 10822–10830] suggesting that this activity might depend on the cellular context of expression of the various KChIPs. These results reveal a new role for KChIP3 in the regulation of Ca2+-regulated secretion and also suggest that the functions of each of the KChIPs may be more specialized than previously appreciated
Data integration in eHealth: a domain/disease specific roadmap
The paper documents a series of data integration workshops held in 2006 at the UK National e-Science Centre, summarizing a range of the problem/solution scenarios in multi-site and multi-scale data integration with six HealthGrid projects using schizophrenia as a domain-specific test case. It outlines emerging strategies, recommendations and objectives for collaboration on shared ontology-building and harmonization of data for multi-site trials in this domain
Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites
Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates
Direct oriented growth of armchair graphene nanoribbons on germanium
Graphene can be transformed from a semimetal into a semiconductor if it is confined into nanoribbons narrower than 10nm with controlled crystallographic orientation and well-defined armchair edges. However, the scalable synthesis of nanoribbons with this precision directly on insulating or semiconducting substrates has not been possible. Here we demonstrate the synthesis of graphene nanoribbons on Ge(001) via chemical vapour deposition. The nanoribbons are self-aligning 3 degrees from the Ge < 110 > directions, are self-defining with predominantly smooth armchair edges, and have tunable width to <10 nm and aspect ratio to >70. In order to realize highly anisotropic ribbons, it is critical to operate in a regime in which the growth rate in the width direction is especially slow, <5 nm h(-1). This directional and anisotropic growth enables nanoribbon fabrication directly on conventional semiconductor wafer platforms and, therefore, promises to allow the integration of nanoribbons into future hybrid integrated circuits
The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction
Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal
muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised
that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12
myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube
hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast
culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation
when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature
myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I
MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area
in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa
and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing
protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to
ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol
evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated
impaired myotube growth observed during glucose restriction
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