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排序方式: 共有250条查询结果,搜索用时 15 毫秒
101.
Root Gorelick 《Biological journal of the Linnean Society. Linnean Society of London》2014,112(3):640-644
Based on the extremely small sizes of their jaw components, I predict that members of the Micrognathozoa will have some of the smallest nuclear genomes of any metazoans or, possibly, even of any free‐living (non‐parasitic) eukaryotes. Micrognathozoan jaws may also be enervated by anucleate neurons. Consistent with the prediction of small genomes, micrognathozoan jaw parts have remarkably small cell nuclei. Identical arguments may apply to other members of the Gnathifera, namely Rotifera and Gnathostomulida. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 640–644. 相似文献
102.
Batoul Y Abdallah Steven D Horne Joshua B Stevens Guo Liu Andrew Y Ying Barbara Vanderhyden Stephen A Krawetz Root Gorelick Henry HQ Heng 《Cell cycle (Georgetown, Tex.)》2013,12(23):3640-3649
Multi-level heterogeneity is a fundamental but underappreciated feature of cancer. Most technical and analytical methods either completely ignore heterogeneity or do not fully account for it, as heterogeneity has been considered noise that needs to be eliminated. We have used single-cell and population-based assays to describe an instability-mediated mechanism where genome heterogeneity drastically affects cell growth and cannot be accurately measured using conventional averages. First, we show that most unstable cancer cell populations exhibit high levels of karyotype heterogeneity, where it is difficult, if not impossible, to karyotypically clone cells. Second, by comparing stable and unstable cell populations, we show that instability-mediated karyotype heterogeneity leads to growth heterogeneity, where outliers dominantly contribute to population growth and exhibit shorter cell cycles. Predictability of population growth is more difficult for heterogeneous cell populations than for homogenous cell populations. Since “outliers” play an important role in cancer evolution, where genome instability is the key feature, averaging methods used to characterize cell populations are misleading. Variances quantify heterogeneity; means (averages) smooth heterogeneity, invariably hiding it. Cell populations of pathological conditions with high genome instability, like cancer, behave differently than karyotypically homogeneous cell populations. Single-cell analysis is thus needed when cells are not genomically identical. Despite increased attention given to single-cell variation mediated heterogeneity of cancer cells, continued use of average-based methods is not only inaccurate but deceptive, as the “average” cancer cell clearly does not exist. Genome-level heterogeneity also may explain population heterogeneity, drug resistance, and cancer evolution. 相似文献
103.
Boehm JS Zhao JJ Yao J Kim SY Firestein R Dunn IF Sjostrom SK Garraway LA Weremowicz S Richardson AL Greulich H Stewart CJ Mulvey LA Shen RR Ambrogio L Hirozane-Kishikawa T Hill DE Vidal M Meyerson M Grenier JK Hinkle G Root DE Roberts TM Lander ES Polyak K Hahn WC 《Cell》2007,129(6):1065-1079
The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery. 相似文献
104.
Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin 下载免费PDF全文
The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke. 相似文献
105.
The relationship of complement consumption to immune fever 总被引:2,自引:0,他引:2
I D Mickenberg R Snyderman R K Root S E Mergenhagen S M Wolff 《Journal of immunology (Baltimore, Md. : 1950)》1971,107(5):1466-1476
106.
107.
Kelly Champagne Akira Shishido Michael J. Root 《The Journal of biological chemistry》2009,284(6):3619-3627
Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves
fusion of viral and cellular membranes and is mediated by structural
transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets
the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational
changes essential for the entry process. To probe the T20 structure-activity
relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled
coil using the 5-Helix design strategy. T20 bound this artificial protein
(denoted 5H-ex) with nanomolar affinity (KD = 30
nm), close to its IC50 concentration (∼3
nm) but much weaker than the affinity of a related inhibitory
C-peptide C37 (KD = 0.0007 nm). T20/C37
competitive binding assays confirmed that T20 interacts with the hydrophobic
groove on the surface of the N-HR coiled coil outside of a deep pocket region
crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C
termini contributed to the inhibitor binding activity. Mutating three aromatic
residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity,
suggesting that these amino acids do not participate in T20 binding to the
gp41 N-HR. The results support recent evidence pointing to a different role
for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A.,
Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278,
21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X.,
Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282,
9612–9620). By contrast, mutations near the T20 N terminus substantially
influenced inhibitor binding strength. When Ile was substituted for Thr in the
second T20 position, a 40-fold increase in binding affinity was measured
(KD = 0.75 nm). The effect of this affinity
enhancement on T20 inhibitory potency varied among different viral strains.
The original T20 and the higher affinity T20 variant had similar potency
against wild type HIV-1. However, the higher affinity T20 variant was
significantly more potent against T20-resistant virus. The findings suggest
that other factors in addition to binding affinity play a role in limiting T20
potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will
be a useful tool to further elucidate mechanistic profiles of C-peptide
inhibitors.The HIV-12 surface
glycoprotein Env promotes viral entry through the fusion of viral and cellular
membranes (3). Env consists of
three gp120 surface subunits and three gp41 transmembrane subunits arranged as
a trimer-of-heterodimers on the virion surface. In the current model of HIV-1
entry, cellular receptor binding to gp120 initiates a series of coordinated
structural transformations that stimulate gp41 to extend and insert its
N-terminal fusion peptide into target cell membranes (see
Fig. 1A)
(4,
5). This high energy extended
intermediate structure ultimately collapses into a trimer-of-hairpins
conformation that juxtaposes the gp41 fusion peptide and transmembrane domain.
Because the fusion peptide and transmembrane domain are inserted in target
cell and viral membranes, formation of the trimer-of-hairpins is proposed to
bring these membranes into the close proximity required for efficient
fusion.Open in a separate windowFIGURE 1.HIV-1 gp41 and its role in viral membrane fusion. A, a
model of HIV-1 entry (46). In
native Env prior to receptor activation, gp41 is held in a metastable
conformation by a canopy of gp120 proteins (green). Receptor binding
to gp120 stimulates gp41 to extend and insert its fusion peptide segment
(red) into the target cell membrane. The N-HR (gray) and
C-HR (blue) regions of the gp41 ectodomain are transiently exposed in
this prehairpin state. Subsequently, gp41 collapses into the
trimer-of-hairpins conformation that brings the gp41 fusion peptides,
transmembrane regions (purple), and their associated membranes into
the close proximity for membrane fusion. The actual disposition of gp120 in
both the prehairpin and trimer-of-hairpins states is uncertain; for clarity,
the protein is omitted in the schematic of the trimer-of-hairpins
conformation. B, a diagram of HIV-1 gp41 identifying its fusion
peptide (FP), N-HR, C-HR, MPER (MP), transmembrane
(TM), and cytoplasmic (cyto) domains. Amino acid sequences
above and below the diagram are derived from the N-HR and C-HR/MPER regions of
EnvHXB2; all but the MPER sequence WNWF (magenta) were
used in the design of 5H-ex. The N-HR and C-HR segments found in the original
5-Helix are boxed in gray and blue, respectively, whereas
the sequences of C37 and T20 are denoted by lines. The side chains of
the C-HR amino acids marked with + pack into the hydrophobic pocket at the C
terminus of the N-HR coiled coil.The core of the trimer-of-hairpins is a bundle of six α-helices
formed by two hydrophobic heptad repeat sequences in the N- and C-terminal
regions of the gp41 ectodomain (N-HR and C-HR, respectively)
(6,
7). In the trimer-of-hairpins,
the N-HR segments from three gp41 ectodomains form a central trimeric coiled
coil, around which the three C-HR segments pack as antiparallel helices into
hydrophobic grooves
(8–11).
In the prehairpin extended conformation, the N-HR and C-HR segments are
unassociated and transiently accessible to inhibitors of HIV-1 entry
(5,
12). Several such inhibitors
are formed from the peptide sequence of the C-HR and adjacent gp41 regions
(4,
6,
13,
14). Denoted C-peptides, they
work in a dominant negative fashion by binding to the exposed N-HR coiled
coil, thereby blocking trimer-of-hairpins formation and inhibiting viral
membrane fusion (4,
15–21).
One C-peptide, T20 (also called enfuvirtide), has shown antiviral activity
in vivo and has been approved for use in the treatment of HIV-1
infection (22,
23).T20 is a 36-amino acid peptide extending from Tyr638 in the
middle of the C-HR to Phe673 in the Trp-rich membrane proximal
external region (MPER) that precedes the gp41 transmembrane domain (residue
numbering is according to the EnvHXB2 sequence; see
Fig. 1B)
(13). In T20, these C-terminal
MPER-derived residues are critical for inhibitory activity, although their
structure and function in the gp41-bound state are currently unknown
(1,
24,
25). A second class of
similarly potent C-peptides includes C34 (residues 628–661) and the
slightly larger C37 (residues 625–661)
(4,
6,
26,
27). These peptides are
derived entirely from the C-HR sequence and thus are shifted in the N-terminal
direction compared with T20 (Fig.
1B). The interactions of C34 and C37 with gp41 are
greatly stabilized by residues Trp628, Trp631, and
Ile635 near the C-HR N terminus
(4). Their bulky hydrophobic
side chains pack into a deep hydrophobic pocket on the surface of the N-HR
coiled coil. T20 lacks these pocket binding residues and their stabilizing
effect. However, T20 does contain bulky hydrophobic residues
(Trp670, Trp672, and Phe673) at its C
terminus that might pack into a similar pocket at the other end of the N-HR
coiled coil.High resolution structures of the gp41 trimer-of-hairpins have aided our
understanding of the mechanism of C-peptide inhibition. These structures have
enabled the design of polypeptides that mimic the gp41 N-HR coiled coil and
bind C34/C37, thereby providing a tool to probe the structure-activity
relationships of the inhibitors
(26,
28–30).
No similar tool is available for investigating T20 inhibition in detail. The
structures of the gp41 trimer-of-hairpins do not include the T20 C terminus (9
residues) nor the gp41 N-terminal segments that putatively interact with it.
Furthermore, gp41 N-HR-derived peptides predicted to interact with T20 are
poorly soluble and difficult to use in solution phase interaction assays
(6). Here we describe the
design of a soluble protein (denoted 5H-ex) that mimics the putative
T20-binding site on the N-HR coiled coil. 5H-ex interacts with T20 with an
equilibrium dissociation constant (KD) of 30
nm, close to the T20 50% inhibitory concentration (IC50)
of 3 nm. Using this protein, we explored the extent to which the N
and C termini of T20 contribute to its binding activity. First, we showed that
the MPER-derived residues at the peptide C terminus do not stabilize the
5H-ex/T20 interaction. Second, we identified an N-terminal substitution that
significantly enhanced T20 binding affinity and improved peptide inhibitory
activity against T20-resistant HIV-1. The results suggest that T20 binding to
the N-HR coiled coil is stabilized primarily by residues derived from the C-HR
and not the MPER. 5H-ex is likely to be a useful tool in probing the
structure-activity relationship of T20. 相似文献
108.
A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen 总被引:22,自引:0,他引:22
Moffat J Grueneberg DA Yang X Kim SY Kloepfer AM Hinkle G Piqani B Eisenhaure TM Luo B Grenier JK Carpenter AE Foo SY Stewart SA Stockwell BR Hacohen N Hahn WC Lander ES Sabatini DM Root DE 《Cell》2006,124(6):1283-1298
To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery. 相似文献
109.
Echeverri CJ Beachy PA Baum B Boutros M Buchholz F Chanda SK Downward J Ellenberg J Fraser AG Hacohen N Hahn WC Jackson AL Kiger A Linsley PS Lum L Ma Y Mathey-Prévôt B Root DE Sabatini DM Taipale J Perrimon N Bernards R 《Nature methods》2006,3(10):777-779
Large-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens. 相似文献
110.
Pumroy RA Nardozzi JD Hart DJ Root MJ Cingolani G 《The Journal of biological chemistry》2012,287(3):2022-2031
The human genome encodes six isoforms of importin α that show greater than 60% sequence similarity and remarkable substrate specificity. The isoform importin α5 can bind phosphorylated cargos such as STAT1 and Epstein-Barr Virus Nuclear Antigen 1, as well as the influenza virus polymerase subunit PB2. In this work, we have studied the interaction of the nucleoporin Nup50 with importin α5. We show that the first 47 residues of Nup50 bind to the C terminus of importin α5 like a "clip," stabilizing the closed conformation of ARM 10. In vitro, Nup50 binds with high affinity either to empty importin α5 or to a preassembled complex of importin α5 bound to the C-terminal domain of the import cargo PB2, resulting in a trimeric complex. By contrast, PB2 can only bind with high affinity to importin α5 in the absence of Nup50. This suggests that Nup50 primary function may not be to actively displace the import cargo from importin α5 but rather to prevent cargo rebinding in preparation for recycling. This is the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor. 相似文献