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51.
Rats were trained to discriminate short or long durations of houselight illumination using a choice procedure. During the test phase of each trial, the left and right levers were presented with an auditory cue above one of them on (cued lever) while the other was off (uncued lever). The auditory cue was presented immediately after sample offset and the levers were inserted after the auditory cue had been presented for 2 s. For half of the rats, the correct response following the short sample was to press the cued lever, while following the long sample, it was to press the uncued lever. This was reversed for the remaining rats. Following acquisition of the discrimination, two different types of delay tests were administered. In the first set, the delay between offset of the sample and onset of the auditory cue was manipulated (Cue Delay Test). In the second set, the delay between onset of the auditory cue and entry of the levers into the chamber was manipulated (Response Delay Test). Cue Delay testing resulted in a choose-long bias at the longer delays. Response Delay testing did not result in a systematic response bias and there was little forgetting over the delay interval. These data suggest that the rats did not stop the internal clock when the nominal sample was offset, but allowed it to keep running until the auditory cue was presented. The data from the Response Delay Test indicate that either a response decision was made based on the clock reading as soon as the auditory cue was presented, or the clock reading itself was retained over the delay with no subjective shortening and little forgetting. 相似文献
52.
Development of a radioactive protein A-based assay for analysis of surface protein expression in gram-positive bacteria. 总被引:1,自引:1,他引:0
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This paper describes an immunochemical method which uses radioactive protein A for the detection and analysis of streptococcal M6 protein epitopes on the surface of recombinant Streptococcus gordonii. With this assay, recombinant S. gordonii cells expressing a portion of the M6 protein on their surfaces show a 75-fold increase in bound radioactivity over cells of the control S. gordonii parental strain. Furthermore, use of the assay to monitor the amount of M6 protein present on the surface of the S. gordonii recombinant during growth in culture demonstrated that expression is highest at late log phase, with the protein being sloughed off during stationary phase. This simple assay allows analysis of surface protein without any protein purification or sophisticated instrumentation. As such, it should be broadly applicable to following the expression of most surface-accessible bacterial proteins. 相似文献
53.
Xinjun Zhang Bryan T. MacDonald Huilan Gao Michael Shamashkin Anthony J. Coyle Robert V. Martinez Xi He 《The Journal of biological chemistry》2016,291(5):2435-2443
The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn2+/Co2+ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates. 相似文献
54.
55.
Chiara Nembrini Jan Kisielow Abdijapar T. Shamshiev Luigi Tortola Anthony J. Coyle Manfred Kopf Benjamin J. Marsland 《The Journal of biological chemistry》2009,284(29):19183-19188
Rip2 (RICK, CARD3) has been identified as a key effector molecule downstream of the pattern recognition receptors, Nod1 and Nod2; however, its mechanism of action remains to be elucidated. In particular, it is unclear whether its kinase activity is required for signaling or for maintaining protein stability. We have investigated the expression level of different retrovirally expressed kinase-dead Rip2 mutants and the role of Rip2 kinase activity in the signaling events that follow Nod1 and Nod2 stimulation. We show that in primary cells expressing kinase-inactive Rip2, protein levels were severely compromised, and stability could not be reconstituted by the addition of a phospho-mimetic mutation in its autophosphorylation site. Consequently, inflammatory cytokine production in response to Nod1 and Nod2 ligands was abrogated both in vitro and in vivo in the absence of Rip2 kinase activity. Our results highlight the central role that Rip2 kinase activity plays in conferring stability to the protein and thus in the preservation of Nod1- and Nod2-mediated innate immune responses.A key step in the initiation of effector immune responses is the recognition of highly conserved molecules expressed by microbial pathogens. The immune system has developed specific receptors that sense these so-called pathogen-associated molecular patterns and initiate appropriate immune responses. One key family of pattern recognition receptors is the Nod-like receptor (NLR)2 family (1–3), of which two members, Nod1 and Nod2, have been implicated in the recognition of bacterial peptidoglycan derivatives released into the cytosol upon bacterial infection (4–6). Several studies have shown that Nod1 plays a role in host defense against invasive pathogens such as Helicobacter pylori and Escherichia coli (7, 8), and Nod2 mutations have been associated with a higher incidence of Crohn disease (9, 10), thus highlighting these NLRs as important regulators of inflammatory immune responses.Rip2, also called CARD3, RICK, or CARDIAK, is a serine/threonine kinase, which was implicated in the induction of NF-κB activation and apoptosis (11–13). Rip2 has been described to be critical for responses against Toll-like receptor ligands such as LPS (14, 15), although findings from recent studies did not support this conclusion (16). Rip2 contains a caspase-recruitment domain (CARD), which mediates interaction with other CARD-containing proteins such as Nod1 and Nod2, in addition to an N-terminal kinase domain and an intermediate domain. Nod1 and Nod2 associate with Rip2 upon peptidoglycan ligation (17) leading to downstream signaling events that culminate in NF-κB and mitogen-activated protein kinase activation (15, 18–20). Recent reports have suggested that the mitogen-activated protein kinase kinase kinase family member TAK1 provides the link between Rip2 and NF-κB activation upon Nod1 and Nod2 stimulation (21–23). However, the exact role of Rip2 and in particular its kinase activity in mediating downstream effector activation in NLR signaling still remains unclear. Notably, in vitro investigations have suggested that Rip2 kinase activity may be dispensable for the induction of immune responses initiated by NLR-ligands (21, 24, 25) and that disruption of Rip2 kinase activity is associated with a loss in protein stability (23); however, such studies utilized protein overexpression in cell lines and are yet to be tested in primary cells or in vivo.In the current investigation we sought to elucidate the role of Rip2 kinase activity in transducing inflammatory signals upon NLR stimulation in vitro and in vivo. To this end, we utilized both Rip2 knock-out (15) and Rip2 kinase-dead knock-in mice (24) in addition to Rip2 deficient primary cells that were retrovirally reconstituted with different kinase-inactive mutants. We show here that in the absence of intact kinase activity, Rip2 protein is not stable and that insertion of a phospho-mimetic mutation is not sufficient to restore stability. Moreover, pharmacological abrogation of Rip2 kinase activity in primary cells similarly leads to destabilization of the molecule. As a consequence, signaling downstream of Nod1 and Nod2 and inflammatory cytokine production is impaired both in vivo and in vitro. Our results highlight Rip2 kinase activity as a central regulator of protein stability and consequently innate immune responses triggered by Nod1 and Nod2 ligands. 相似文献
56.
Kenshi Yamasaki Jun Muto Kristen R. Taylor Anna L. Cogen David Audish John Bertin Ethan P. Grant Anthony J. Coyle Amirhossein Misaghi Hal M. Hoffman Richard L. Gallo 《The Journal of biological chemistry》2009,284(19):12762-12771
Inflammation under sterile conditions is a key event in autoimmunity and
following trauma. Hyaluronan, a glycosaminoglycan released from the
extracellular matrix after injury, acts as an endogenous signal of trauma and
can trigger chemokine release in injured tissue. Here, we investigated whether
NLRP3/cryopyrin, a component of the inflammasome, participates in the
inflammatory response to injury or the cytokine response to hyaluronan. Mice
with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in
response to sterile injuries but had decreased inflammation and release of
interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to
macrophages derived from cryopyrin-deficient mice increased release of Cxcl2
but did not increase IL-1β release. To define the mechanism of
hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan
recognition process were studied in detail. IL-1β release was inhibited
in peritoneal macrophages derived from CD44-deficient mice, in an MH-S
macrophage cell line treated with antibodies to CD44, or by inhibitors of
lysosome function. The requirement for CD44 binding and hyaluronan
internalization could be bypassed by intracellular administration of
hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed
macrophages. Therefore, the action of CD44 and subsequent hyaluronan
catabolism trigger the intracellular cryopyrin → IL-1β pathway.
These findings support the hypothesis that hyaluronan works through IL-1β
and the cryopyrin system to signal sterile inflammation.Inflammation, as defined by changes in vascular permeability and leukocyte
recruitment, is an essential step for the control of microbial invasion.
Specific microbial products trigger this process through a diverse array of
innate immune pattern recognition receptors. However, an inflammatory response
independent of infection is also an important process for maintenance of
biological homeostasis. For example, normal wound healing requires a
controlled inflammatory response to enable the recruitment of monocytes and
the release of growth factors required for repair. This response can occur in
the absence of microbial stimuli. Furthermore, inflammation and the release of
proinflammatory mediators is also associated with many diseases such as
rheumatoid arthritis and Crohn disease
(1). These diseases are not
well understood in terms of their triggers but rather are described by the
subsequent release of proinflammatory mediators. Identification of the
triggers of sterile inflammation represents an important goal with immediate
diagnostic and therapeutic significance.Recent work has begun to elucidate pathways of inflammation that occur in
the absence of microbial stimuli. Stress signals such as heat-shock proteins,
intracellular components of necrotic cells not normally seen by immune cells,
and components of the extracellular matrix have all been implicated as
endogenous triggers of injury
(2–4).
Among this group is the glycosaminoglycan hyaluronan
(HA),6 an important
structural component of the extracellular matrix that is also a common
component of bacterial surfaces. HA is synthesized at the cell surface and
typically exists as a high molecular mass polymer greater than 106
Da and composed of repeating disaccharide units of
N-acetylglucosamine and glucuronic acid
(5,
6). Unlike other
glycosaminoglycans such as heparan sulfate or chondroitin sulfates that encode
specific activity by use of a diverse disaccharide sequence, HA is not
sulfated or epimerized, and only changes in HA size, concentration, and
location affect function.We have previously developed murine models of sterile injury to identify
the innate elements that recognize and mediate sterile inflammation
(7). Our results demonstrated
that (a) the initiation of a sterile intrinsic inflammatory process
is dependent on TLR4 activation, (b) sterile injury induces HA
accumulation at the injured site, and (c) sterile intrinsic
inflammation resembles signaling events that are activated by HA. Furthermore,
we have defined a novel alternative recognition complex for HA that involves
TLR4, MD-2, and CD44 (7). Taken
together with other work associating HA and innate pattern recognition
(4,
8–10),
these observations have provided new insight into mechanisms responsible for
sterile inflammation.Recently, the NLR (nucleotide-binding domain and leucine rich
repeat-containing) family has been extensively analyzed as a group of
intracellular pattern recognition receptors
(11). NLRs have a leucine-rich
repeat that recognizes pathogen-associated molecular patterns including
bacterial cell wall components and viral nucleic acids. NOD2 and NLR family,
pyrin containing 3 (NLRP3)/cryopyrin are two of the best
characterized NLRs. NOD2 recognizes the bacterial peptidoglycan-derived
molecule muramyl dipeptide and activates the NF-κB pathway to induce
inflammatory responses (12).
Mutations of the NOD2 gene were identified in individuals with
chronic inflammatory disorders such as Crohn disease
(13,
14) and Blau syndrome
(15). Mouse knockin mutants of
NOD2, which have the same mutation in NOD2 as human patients
with Crohn disease, showed elevated proinflammatory cytokines following
muramyl dipeptide challenge or dextran sodium sulfate-induced bowel
inflammation (16).
NLRP3, also known as cyropyrin, CIAS1, NALP3, PYPAF1, forms
an “inflammasome” with ASC (apoptosis-associated speck-like
protein containing a CARD) and caspase-1 to convert pro-IL-1β to active
IL-1β (17). Mutations in
NLRP3 were identified in individuals with familial cold
autoinflammatory syndrome (FCAS), Muckle-Wells syndrome, and neonatal onset
multisystem inflammatory disease
(18–20).
These individuals have recurrent or chronic inflammatory symptoms, including
fever, arthritis, and a urticaria-like eruption characterized by neutrophilic
infiltration. In FCAS, symptoms can be elicited by cold provocation by a
mechanism that appears to be mediated through the skin
(15,
21).Because disorders associated with mutations in NLRP3 are examples
of inflammation under sterile conditions and HA has been shown to be a trigger
of sterile inflammation, we sought to further understand the mechanism of the
response to HA by examining the role of cryopyrin during injury and after
exposure to HA. Our results show that cryopyrin and IL-1β are integral to
sterile inflammation and the response to HA. These observations provide new
insight into the function of HA as a “danger signal” of
injury. 相似文献
57.
58.
Nick Wheelhouse Christopher Coyle Peter G. Barlow Stephen Mitchell Gilbert Greub Tim Baszler Mick T. Rae David Longbottom 《PloS one》2014,9(7)
Background
Waddlia chondrophila (W. chondrophila) is an emerging abortifacient organism which has been identified in the placentae of humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial abortifacients, such as Chlamydia abortus (C. abortus). This study investigates the growth of the organism and its effects upon pro-inflammatory cytokine expression in a ruminant placental cell line which we have previously utilised in a model of C. abortus pathogenicity.Methodology/Principal Findings
Using qPCR, fluorescent immunocytochemistry and electron microscopy, we characterised the infection and growth of W. chondrophila within the ovine trophoblast AH-1 cell line. Inclusions were visible from 6 h post-infection (p.i.) and exponential growth of the organism could be observed over a 60 h time-course, with significant levels of host cell lysis being observed only after 36 h p.i. Expression of CXCL8, TNF-α, IL-1α and IL-1β were determined 24 h p.i. A statistically significant response in the expression of CXCL8, TNF-α and IL-1β could be observed following active infection with W. chondrophila. However a significant increase in IL-1β expression was also observed following the exposure of cells to UV-killed organisms, indicating the stimulation of multiple innate recognition pathways.Conclusions/Significance
W. chondrophila infects and grows in the ruminant trophoblast AH-1 cell line exhibiting a complete chlamydial replicative cycle. Infection of the trophoblasts resulted in the expression of pro-inflammatory cytokines in a dose-dependent manner similar to that observed with C. abortus in previous studies, suggesting similarities in the pathogenesis of infection between the two organisms. 相似文献59.
Sarah E. Knowles Gill Toms Caroline Sanders Penny Bee Karina Lovell Stefan Rennick-Egglestone David Coyle Catriona M. Kennedy Elizabeth Littlewood David Kessler Simon Gilbody Peter Bower 《PloS one》2014,9(1)
Objective
Computerised therapies play an integral role in efforts to improve access to psychological treatment for patients with depression and anxiety. However, despite recognised problems with uptake, there has been a lack of investigation into the barriers and facilitators of engagement. We aimed to systematically review and synthesise findings from qualitative studies of computerised therapies, in order to identify factors impacting on engagement.Method
Systematic review and meta-synthesis of qualitative studies of user experiences of computer delivered therapy for depression and/or anxiety.Results
8 studies were included in the review. All except one were of desktop based cognitive behavioural treatments. Black and minority ethnic and older participants were underrepresented, and only one study addressed users with a co-morbid physical health problem. Through synthesis, we identified two key overarching concepts, regarding the need for treatments to be sensitive to the individual, and the dialectal nature of user experience, with different degrees of support and anonymity experienced as both positive and negative. We propose that these factors can be conceptually understood as the ‘non-specific’ or ‘common’ factors of computerised therapy, analogous to but distinct from the common factors of traditional face-to-face therapies.Conclusion
Experience of computerised therapy could be improved through personalisation and sensitisation of content to individual users, recognising the need for users to experience a sense of ‘self’ in the treatment which is currently absent. Exploiting the common factors of computerised therapy, through enhancing perceived connection and collaboration, could offer a way of reconciling tensions due to the dialectal nature of user experience. Future research should explore whether the findings are generalisable to other patient groups, to other delivery formats (such as mobile technology) and other treatment modalities beyond cognitive behaviour therapy. The proposed model could aid the development of enhancements to current packages to improve uptake and support engagement. 相似文献60.
C-peptide inhibits leukocyte-endothelium interaction in the microcirculation during acute endothelial dysfunction. 总被引:3,自引:0,他引:3
C-peptide is a cleavage product that comes from processing proinsulin to insulin that induces nitric oxide (NO) -mediated vasodilation. NO modulates leukocyte-endothelium interaction. We hypothesized that C-peptide might inhibit leukocyte-endothelium interaction via increased release of endothelial NO. Using intravital microscopy of the rat mesentery, we measured leukocyte-endothelium interactions after administration of C-peptide to the rat. Superfusion of the rat mesentery with either thrombin or L-NAME consistently and significantly increased the number of rolling, adhering, and transmigrated leukocytes. C-peptide significantly attenuated either thrombin- or L-NAME-induced leukocyte-endothelium interactions in rat mesenteric venules. A control scrambled sequence of C-peptide characterized by the same amino acid composition in a randomized sequence failed to inhibit leukocyte-endothelium interactions. These effects of C-peptide were associated with decreased surface expression of the cell adhesion molecules P-selectin and ICAM-1 on the microvascular endothelium. Endothelial nitric oxide synthase (eNOS) mRNA levels were increased in rats injected with C-peptide. This enhanced eNOS expression was associated with a marked increase in basal NO release from the aorta of C-peptide-treated rats. We conclude that C-peptide is a potent inhibitor of leukocyte-endothelium interaction and that this effect is specifically related to inhibition of endothelial cell adhesion molecules via maintenance of NO release from the vascular endothelium. 相似文献