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91.
Autoimmunity and the microbiome: T‐cell receptor mimicry of “self” and microbial antigens mediates self tolerance in holobionts 下载免费PDF全文
Robert Root‐Bernstein 《BioEssays : news and reviews in molecular, cellular and developmental biology》2016,38(11):1068-1083
92.
Copper iodide staining which can detect protein levels as low as 100-150 pg/mm2 on nitrocellulose membranes is described. The staining is quantitative as measured by densitometry. Staining is complete within 5 min and may be removed by washing the membrane for 15 min without loss of immunoreactivity. The stain utilizes a reddish-brown precipitate of copper iodide in highly alkaline conditions. Because of its high sensitivity, convenience, and low cost, this stain may be more practical than amido black or gold- and silver-based stains for most laboratory purposes. 相似文献
93.
94.
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
95.
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
96.
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
97.
L Alonso EC Souza MV Oliveira LFE do Nascimento PMS Dantas 《Biology of sport / Institute of Sport》2014,31(4):267-270
The objective of this study was to evaluate the genetic and environmental contribution to variation in aerobic power in monozygotic (MZ) and dizygotic (DZ) twins. The sample consisted of 20 MZ individuals (12 females and 8 males) and 16 DZ individuals (12 females and 4 males), aged from 8 to 26 years, residents in Natal, Rio Grande do Norte. The twins were assessed by a multistage fitness test. The rate of heritability found for aerobic power was 77%. Based on the results, the estimated heritability was largely responsible for the differences in aerobic power. This implies that such measures are under strong genetic influence. 相似文献
98.
99.
Domenico Sambataro Gianluca Sambataro Eleonora Zaccara Wanda Maglione Riccardo Polosa Antonella MV Afeltra Claudio Vitali Nicoletta Del Papa 《Arthritis research & therapy》2014,16(5)
Introduction
Nailfold videocapillaroscopy (NVC) in systemic sclerosis (SSc) is a procedure commonly used for patient classification and subsetting, but not to define disease activity (DA). This study aimed to evaluate whether the number of micro-haemorrhages (MHE), micro-thrombosis (MT), giant capillaries (GC), and normal/dilated capillaries (Cs) in NVC could predict DA in SSc.Methods
Eight-finger NVC was performed in 107 patients with SSc, and the total number of MHE/MT, GC, and the mean number of Cs were counted and defined as number of micro-haemorrhages (NEMO), GC and Cs scores, respectively. The European Scleroderma Study Group (ESSG) index constituted the gold standard for DA assessment, and scores ≥3.5 and =3 were considered indicative of high and moderate activity, respectively.Results
NEMO and GC scores were positively correlated with ESSG index (R = 0.65, P <0.0001, and R = 0.47, P <0.0001, respectively), whilst Cs score showed a negative correlation with that DA index (R = −0.30, P <0.001). The area under the curve (AUC) of receiver operating characteristic plots, obtained by NEMO score sensitivity and specificity values in classifying patients with ESSG index ≥3.5, was significantly higher than the corresponding AUC derived from either GC or Cs scores (P <0.03 and P <0.0006, respectively). A modified score, defined by the presence of a given number of MHE/MT and GC, had a good performance in classifying active patients (ESSG index ≥3, sensitivity 95.1%, specificity 84.8%, accuracy 88.7%).Conclusions
MHE/MT and GC appear to be good indicators of DA in SSc, and enhances the role of NVC as an easy technique to identify active patients. 相似文献100.
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