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921.
Phillip A. Wilmarth Michael A. Riviere Larry L. David 《Journal of ocular biology, diseases, and informatics》2009,2(4):223-234
Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses. 相似文献
922.
The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H2) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H2 production at various growth temperatures, (2) investigate the effect of oxygen (O2) on H2 production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H2 production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H2, 2 mol CO2, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H2 production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H2 through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
923.
Hao-Chieh Chiu Shilpa Soni Samuel K Kulp Heather Curry Dasheng Wang John S Gunn Larry S Schlesinger Ching-Shih Chen 《Journal of biomedical science》2009,16(1):110
Background
Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.Methods and results
Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis (type A strain, Schu S4) and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria.Conclusion
Together, AR-12 represents a proof-of-principle that intracellular F. tularensis can be eradicated by small-molecule agents that target innate immunity. 相似文献924.
Joanne A. Labate Larry D. Robertson Feinan Wu Steven D. Tanksley Angela M. Baldo 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,118(5):1005-1014
Because cultivated tomato (Solanum lycopersicum L.) is low in genetic diversity, public, verified single nucleotide polymorphism (SNP) markers within the species are in
demand. To promote marker development we resequenced approximately 23 kb in a diverse set of 31 tomato lines including TA496.
Three classes of markers were sampled: (1) 26 expressed-sequence tag (EST), all of which were predicted to be polymorphic
based on TA496, (2) 14 conserved ortholog set II (COSII) or unigene, and (3) ten published sequences, composed of nine fruit
quality genes and one anonymous RFLP marker. The latter two types contained mostly noncoding DNA. In total, 154 SNPs and 34
indels were observed. The distributions of nucleotide diversity estimates among marker types were not significantly different
from each other. Ascertainment bias of SNPs was evaluated for the EST markers. Despite the fact that the EST markers were
developed using SNP prediction within a sample consisting of only one TA496 allele and one additional allele, the majority
of polymorphisms in the 26 EST markers were represented among the other 30 tomato lines. Fifteen EST markers with published
SNPs were more closely examined for bias. Mean SNP diversity observations were not significantly different between the original
discovery sample of two lines (53 SNPs) and the 31 line diversity panel (56 SNPs). Furthermore, TA496 shared its haplotype
with at least one other line at 11 of the 15 markers. These data demonstrate that public EST databases and noncoding regions
are a valuable source of unbiased SNP markers in tomato.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such
use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural
Research Service of any product or service to the exclusion of others that may be suitable. 相似文献
925.
Metapopulation extinction risk is the probability that all local populations are simultaneously extinct during a fixed time frame. Dispersal may reduce a metapopulation’s extinction risk by raising its average per-capita growth rate. By contrast, dispersal may raise a metapopulation’s extinction risk by reducing its average population density. Which effect prevails is controlled by habitat fragmentation. Dispersal in mildly fragmented habitat reduces a metapopulation’s extinction risk by raising its average per-capita growth rate without causing any appreciable drop in its average population density. By contrast, dispersal in severely fragmented habitat raises a metapopulation’s extinction risk because the rise in its average per-capita growth rate is more than offset by the decline in its average population density. The metapopulation model used here shows several other interesting phenomena. Dispersal in sufficiently fragmented habitat reduces a metapopulation’s extinction risk to that of a constant environment. Dispersal between habitat fragments reduces a metapopulation’s extinction risk insofar as local environments are asynchronous. Grouped dispersal raises the effective habitat fragmentation level. Dispersal search barriers raise metapopulation extinction risk. Nonuniform dispersal may reduce the effective fraction of suitable habitat fragments below the extinction threshold. Nonuniform dispersal may make demographic stochasticity a more potent metapopulation extinction force than environmental stochasticity. 相似文献
926.
Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities 总被引:2,自引:0,他引:2
Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic
bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants.
Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol
and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable
of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera
Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated
that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for
phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for
catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity
provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the
Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically
distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways
they employ for phenol degradation. 相似文献
927.
Larry R. Masterson Nadia Bortone Tao Yu Kim N. Ha Ece C. Gaffarogullari Oanh Nguyen Gianluigi Veglia 《Protein expression and purification》2009,64(2):231-236
Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI5–24). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His6 tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis. 相似文献
928.
929.
Lina Du Robert W. Hickey H��lya Bayir Simon C. Watkins Vladimir A. Tyurin Fengli Guo Patrick M. Kochanek Larry W. Jenkins Jin Ren Greg Gibson Charleen T. Chu Valerian E. Kagan Robert S. B. Clark 《The Journal of biological chemistry》2009,284(4):2383-2396
Sex-dependent differences in adaptation to famine have long been
appreciated, thought to hinge on female versus male preferences for
fat versus protein sources, respectively. However, whether these
differences can be reduced to neurons, independent of typical nutrient depots,
such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A
vital adaptation to starvation is autophagy, a mechanism for recycling amino
acids from organelles and proteins. Here we show that segregated neurons from
males in culture are more vulnerable to starvation than neurons from females.
Nutrient deprivation decreased mitochondrial respiration, increased
autophagosome formation, and produced cell death more profoundly in neurons
from males versus females. Starvation-induced neuronal death was
attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7
knockdown using small interfering RNA; or l-carnitine, essential
for transport of fatty acids into mitochondria, all more effective in neurons
from males versus females. Relative tolerance to nutrient deprivation
in neurons from females was associated with a marked increase in triglyceride
and free fatty acid content and a cytosolic phospholipase A2-dependent
increase in formation of lipid droplets. Similar sex differences in
sensitivity to nutrient deprivation were seen in fibroblasts. However,
although inhibition of autophagy using Atg7 small interfering RNA
inhibited cell death during starvation in neurons, it increased cell death in
fibroblasts, implying that the role of autophagy during starvation is both
sex- and tissue-dependent. Thus, during starvation, neurons from males more
readily undergo autophagy and die, whereas neurons from females mobilize fatty
acids, accumulate triglycerides, form lipid droplets, and survive longer.Sex-dependent differences in adaptation to famine have long been
appreciated (1,
2), thought to hinge on a
female preference for fat sources, in contrast to a male preference for
protein sources (3). Fatty acid
metabolism is different between sexes normally
(4) and under conditions of
starvation (1,
2). During exercise, in
addition to increases in carbohydrate requirement, men increase their need for
amino acids, whereas women increase mobilization of fat
(5). Furthermore, sex-dependent
responses to nutritional stress associated with either self-induced weight
loss or illness-related cachexia also exist
(6,
7).An important adaptation to starvation is autophagy (autophagy-associated
proteins, abbreviated ATG). Classic, starvation-induced autophagy is initiated
by nutrient and amino acid deprivation, glucagon, and cAMP
(8,
9). ATG7, a ubiquitin E1-like
enzyme, is essential for autophagy, with phosphorylation of preautophagosomal
membranes, formation of ATG12-ATG5 complexes, and processing of ATG8/LC3
(microtubule-associated protein light chain-3) as other crucial steps in this
process (10).
Starvation-induced autophagy is regulated by class III phosphatidylinositol
3-kinase and the Bcl-2-interacting partner, Beclin-1
(11). The autophagosomes then
engulf cytoplasmic material and/or organelles, such as mitochondria, the
latter sometimes referred to as “mitophagy,” disassembling large
proteins and organelles to recycle amino acids and other nutrients, an
important response to starvation
(12).It is unknown whether starvation can induce autophagy in the brain;
however, there is evidence that critical starvation can result in brain
atrophy in humans. It has been reported that ∼30% of people during a
prolonged hunger strike (mean of 199 days) will show brain tissue loss
(13), and brain shrinkage in
patients with anorexia nervosa is well documented
(14,
15). Although 48 h of food
deprivation does not produce detectable autophagy in brains from mice
(16), the aforementioned
reports are consistent with long durations of starvation as a bona
fide stimulus for autophagy in brain. There are recent studies suggesting
that other stimuli can induce autophagy in the brain, such as trauma
(17) and ischemia
(18), and that autophagy may
contribute to neuronal death. There is also evidence for autophagy in the
human brain after trauma and critical illness
(19), which probably includes
both elements of malnutrition and systemic stress. A potential role for brain
atrophy as a contributor to neurological morbidity in the critically ill and
injured is an emerging topic
(20). 相似文献
930.
The ubiquitously expressed reduced folate carrier (RFC) is the major
transport system for folate cofactors in mammalian cells and tissues. Previous
considerations of RFC structure and mechanism were based on the notion that
RFC monomers were sufficient to mediate transport of folate and antifolate
substrates. The present study examines the possibility that human RFC (hRFC)
exists as higher order homo-oligomers. By chemical cross-linking, transiently
expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional
cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting,
hRFC species with molecular masses of hRFC homo-oligomers were identified.
Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were
co-immunoprecipitated from both membrane particulate and surface-enriched
membrane fractions, indicating that oligomeric hRFC is expressed at the cell
surface. By co-expression of wild type and inactive mutant S138C hRFCs,
combined with surface biotinylation and confocal microscopy, a
dominant-negative phenotype was demonstrated involving greatly decreased cell
surface expression of both mutant and wild type carrier caused by impaired
intracellular trafficking. For another hRFC mutant (R373A), expression of
oligomeric wild type-mutant hRFC was accompanied by a significant and
disproportionate loss of wild type activity unrelated to the level of surface
carrier. Collectively, our results demonstrate the existence of hRFC
homo-oligomers. They also establish the likely importance of these higher
order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the
synthesis of nucleotide precursors, serine, and methionine in one-carbon
transfer reactions (1). Because
mammals cannot synthesize folates de novo, cellular uptake of these
derivatives is essential for cell growth and tissue regeneration
(2,
3). Folates are hydrophilic
anionic molecules that do not cross biological membranes by diffusion alone,
so it is not surprising that sophisticated membrane transport systems have
evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier
(RFC)2 is widely
considered to be the major transport system for folate co-factors in mammalian
cells and tissues (3,
4). RFC plays a generalized
role in folate transport and provides specialized tissue functions such as
transport across the basolateral membrane of renal proximal tubules
(5), transplacental transport
of folates (6), and folate
transport across the blood-brain barrier
(7), although the contribution
of RFC to intestinal absorption of folates remains controversial
(8,
9). Loss of RFC expression or
function portends potentially profound physiologic and developmental
consequences associated with folate deficiency
(10). RFC is also a major
transporter of antifolate drugs used for cancer chemotherapy such as
methotrexate (Mtx), pemetrexed, and raltitrexed
(4). Loss of RFC expression or
synthesis of mutant RFC protein in tumor cells results in antifolate
resistance caused by incomplete inhibition of cellular enzyme targets and low
levels of antifolate substrate for polyglutamate synthesis
(4,
11).Reflecting its particular physiologic and pharmacologic importance,
interest in RFC structure and function has been high. Since 1994, when murine
RFC was first cloned (12),
application of state-of-the-art molecular biology and biochemistry methods for
characterizing polytopic membrane proteins has led to a progressively detailed
picture of the molecular structure of the carrier, including its membrane
topology, N-glycosylation, functionally or structurally important
domains and amino acids, and packing of α-helix transmembrane domains
(TMDs) (4,
13). Although no crystal
structure for RFC has yet been reported, a detailed homology model for human
RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol
3-phosphate/inorganic phosphate antiporter GlpT was generated
(13,
14) that permits testing of
hypotheses related to hRFC structure and mechanism in a manner not previously
possible.Considerations of hRFC structure and mechanism to date have all been based
on the notion that a single 591-amino acid hRFC molecule is sufficient to
mediate concentrative uptake of folate and antifolate substrates. However, a
growing literature suggests that quaternary structure involving the formation
of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly
an important feature of the structure and function of many membrane
transporters
(15-18).
For major facilitator superfamily proteins, both monomeric (e.g.
LacY, GlpT, UhpT, and GLUT3)
(19-22)
and oligomeric (e.g. LacS, AE1, GLUT1, and TetA)
(23-28)
structures have been reported, establishing the lack of a clear structural
consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a
homo-oligomeric species composed of multiple hRFC monomers. Based on results
with an assortment of biochemical methods with wt and a collection of mutant
hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but
also establish the probable importance of these higher order structures to
intracellular trafficking and carrier function. 相似文献