共查询到20条相似文献,搜索用时 31 毫秒
1.
Egger S Chaikuad A Klimacek M Kavanagh KL Oppermann U Nidetzky B 《The Journal of biological chemistry》2012,287(3):2119-2129
Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD+-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a thiohemiacetal enzyme intermediate in the course of the first oxidation step. The thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu161) by an incompetent analog (Gln161) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD+, we succeeded in trapping a thiohemiacetal enzyme intermediate and determined its structure at 2.3 Å resolution. Cys276 was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD+. The proposed catalytic mechanism of human UGDH involves Lys220 as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp280 deprotonates Cys276 to function as an aldehyde trap and also provides oxyanion stabilization. Glu161 is the Brønsted base catalytically promoting the thioester hydrolysis. 相似文献
2.
Zijun Xiao Chuanjuan Lv Chao Gao Jiayang Qin Cuiqing Ma Zhen Liu Peihai Liu Lixiang Li Ping Xu 《PloS one》2010,5(1)
Background
The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.Methodology/Principal Findings
Simultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.Conclusions/Significance
A recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis. 相似文献3.
Settles M Etzrodt M Kosanke K Schiemann M Zimmermann A Meier R Braren R Huber A Rummeny EJ Weissleder R Swirski FK Wildgruber M 《PloS one》2011,6(10):e25197
Objective
To explore the capacity of human CD14+CD16++ and CD14++CD16- monocytes to phagocyte iron-oxide nanoparticles in vitro.Methods
Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.Results
Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14++CD16- monocytes displayed a significantly higher uptake compared to CD14+CD16++ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14+CD16++ and CD14++CD16- monocytes differently.Conclusion
Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties. 相似文献4.
Huh JW Robinson RC Lee HS Lee JI Heo YS Kim HT Lee HJ Cho SW Choe H 《Protein and peptide letters》2006,13(8):859-862
UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer. 相似文献
5.
Objectives
To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.Results
In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD+. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.Conclusions
Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.6.
Lai KK Stogios PJ Vu C Xu X Cui H Molloy S Savchenko A Yakunin A Gonzalez CF 《PloS one》2011,6(8):e23269
Background
Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2.Methodology/Principal Findings
We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser106, His225, and Asp197, while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank.Conclusions
The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases. 相似文献7.
Hui Sun Lee 《Archives of biochemistry and biophysics》2009,486(1):35-23887
UDP-glucose dehydrogenase (UGDH) is an enzyme catalyzing the conversion of UDP-glucose to UDP-glucuronic acid. Site-directed mutagenesis studies have revealed that human UGDH (hUGDH) has distinct oligomeric states that vary with different point mutations. In this study we have investigated how the changes in the oligomer-forming propensity may be involved in the thermal motion of wild-type hUGDH and its mutants, using normal mode analysis (NMA). Our results show that the perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. In particular, structural stability at the dimeric interface is necessary to retain the hexameric structure of hUGDH. 相似文献
8.
Christopher G. Langendorf Trevor L. G. Key Gustavo Fenalti Wan-Ting Kan Ashley M. Buckle Tom Caradoc-Davies Kellie L. Tuck Ruby H. P. Law James C. Whisstock 《PloS one》2010,5(2)
Background
In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.Methodology/Principal Findings
Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Conclusions/Significance
Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease. 相似文献9.
Rationale
In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).Objective
To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.Methods
Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.Results
In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.Conclusion
TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves. 相似文献10.
Waehre A Halvorsen B Yndestad A Husberg C Sjaastad I Nygård S Dahl CP Ahmed MS Finsen AV Reims H Louch WE Hilfiker-Kleiner D Vinge LE Roald B Attramadal H Lipp M Gullestad L Aukrust P Christensen G 《PloS one》2011,6(4):e18668
Rationale
Inflammatory mechanisms have been suggested to play a role in the development of heart failure (HF), but a role for chemokines is largely unknown. Based on their role in inflammation and matrix remodeling in other tissues, we hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling during HF.Objective
We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5 in cardiac pathophysiology leading to HF.Methods and Results
Mice harboring a systemic knockout of the CXCR5 (CXCR5−/−) displayed increased mortality during a follow-up of 80 days after aortic banding (AB). Following three weeks of AB, CXCR5−/− developed significant left ventricular (LV) dilatation compared to wild type (WT) mice. Microarray analysis revealed altered expression of several small leucine-rich proteoglycans (SLRPs) that bind to collagen and modulate fibril assembly. Protein levels of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced in AB CXCR5−/− compared to AB WT mice. Electron microscopy revealed loosely packed extracellular matrix with individual collagen fibers and small networks of proteoglycans in AB CXCR5−/− mice. Addition of CXCL13 to cultured cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF, we observed increased myocardial levels of CXCR5 and SLRPs, which was reversed following LV assist device treatment.Conclusions
Lack of CXCR5 leads to LV dilatation and increased mortality during pressure overload, possibly via lack of an increase in SLRPs. This study demonstrates a critical role of the chemokine CXCL13 and CXCR5 in survival and maintaining of cardiac structure upon pressure overload, by regulating proteoglycans essential for correct collagen assembly. 相似文献11.
Lee ST Chu K Jung KH Kim JM Moon HJ Bahn JJ Im WS Sunwoo J Moon J Kim M Lee SK Roh JK 《PloS one》2012,7(4):e35713
Background
Activated endothelial cells release plasma membrane submicron vesicles expressing CD62E (E-selectin) into blood, known as endothelial microparticles (EMPs). We studied whether the levels of endothelial microparticles expressing CD62E+, CD31+/Annexin-V+, or CD31+/CD42− predict cardiovascular outcomes in patients with stroke history.Methods/Principal Findings
Patients with stroke history at least 3 months prior to enrolment were recruited. Peripheral blood EMP levels were measured by flow cytometry. Major cardiovascular events and death were monitored for 36 months. Three hundred patients were enrolled, of which 298 completed the study according to protocol. Major cardiovascular events occurred in 29 patients (9.7%). Nine patients died, five from cardiovascular causes. Cumulative event-free survival rates were lower in patients with high levels of CD62E+ microparticles. Multivariate Cox regression analysis adjusted for cardiovascular risk factors, medications and stroke etiologic groups showed an association between a high CD62E+ microparticle level and a risk of major cardiovascular events and hospitalization. Levels of other kinds of EMPs expressing CD31+/Annexin-V+ or CD31+/CD42− markers were not predictive of cardiovascular outcomes.Conclusion
A high level of CD62E+ microparticles is associated with cardiovascular events in patients with stroke history, suggesting that the systemic endothelial activation increases the risk for cardiovascular morbidities. 相似文献12.
Gutiérrez C Díaz L Vallejo A Hernández-Novoa B Abad M Madrid N Dahl V Rubio R Moreno AM Dronda F Casado JL Navas E Pérez-Elías MJ Zamora J Palmer S Muñoz E Muñoz-Fernández MÁ Moreno S 《PloS one》2011,6(12):e27864
Objective
The primary objective was to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy.Methods
We performed an open-label pilot phase II clinical trial involving chronically HIV-1-infected patients receiving stable antiretroviral therapy whose regimen was intensified with 48 weeks of maraviroc therapy. We analyzed the latent reservoir, the residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation.Results
Overall a non significant reduction in the size of the latent reservoir was found (p = 0.068). A mean reduction of 1.82 IUPM was observed in 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification. No effect on plasma residual viremia was observed. Unexpectedly, all the patients had detectable 2LTR DNA circles at week 24, while none of them showed those circles at the end of the study. No changes were detected in CD4+ or CD8+ counts, although a significant decrease was found in the proportion of HLA-DR+/CD38+ CD4+ and CD8+ T-cells. LPS and sCD14 levels increased.Conclusions
Intensification with MVC was associated with a trend to a decrease in the size of the latent HIV-1 reservoir in memory T cells. No impact on residual viremia was detected. Additional studies with larger samples are needed to confirm the results.Trial Registration
ClinicalTrials.gov NCT00795444相似文献13.
Cooper DL Martin SG Robinson JI Mackie SL Charles CJ Nam J;YEAR Consortium Isaacs JD Emery P Morgan AW 《PloS one》2012,7(1):e28918
Objective
The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.Methods
FcγRIIIa/CD16 expression on CD14low and CD14++ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.Results
Increased FcγRIIIa/CD16 expression on CD14++ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14++ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14++ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.Conclusion
Increased FcγRIIIa/CD16 expression on CD14++ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy. 相似文献14.
Sasson SC Zaunders JJ Seddiki N Bailey M McBride K Koelsch KK Merlin KM Smith DE Cooper DA Kelleher AD 《PloS one》2012,7(2):e31148
Aim
HIV infection is associated with distortion of T-cell homeostasis and the IL-7/IL7R axis. Progressive infection results in loss of CD127+132− and gains in CD127−132+ CD4+ and CD8+ T-cells. We investigated the correlates of loss of CD127 from the T-cell surface to understand mechanisms underlying this homeostatic dysregulation.Methods
Peripheral and cord blood mononuclear cells (PBMCs; CBMC) from healthy volunteers and PBMC from patients with HIV infection were studied. CD127+132−, CD127+132+ and CD127−132+ T-cells were phenotyped by activation, differentiation, proliferation and survival markers. Cellular HIV-DNA content and signal-joint T-cell receptor excision circles (sjTRECs) were measured.Results
CD127+132− T-cells were enriched for naïve cells while CD127−132+ T-cells were enriched for activated/terminally differentiated T-cells in CD4+ and CD8+ subsets in health and HIV infection. HIV was associated with increased proportions of activated/terminally differentiated CD127−132+ T-cells. In contrast to CD127+132− T-cells, CD127−132+ T-cells were Ki-67+Bcl-2low and contained increased levels of HIV-DNA. Naïve CD127+132− T-cells contained a higher proportion of sjTRECs.Conclusion
The loss of CD127 from the T-cell surface in HIV infection is driven by activation of CD127+132− recent thymic emigrants into CD127−132+ activated/terminally differentiated cells. This process likely results in an irreversible loss of CD127 and permanent distortion of T-cell homeostasis. 相似文献15.
We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells. 相似文献
16.
Chun CZ Remadevi I Schupp MO Samant GV Pramanik K Wilkinson GA Ramchandran R 《PloS one》2011,6(2):e14732
Background
Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.Methodology/Principal Findings
In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp + cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli + etsrp + hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp + cell numbers at the LPM.Conclusions/Significance
This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish. 相似文献17.
Objectives
Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.Methods
Murine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis.Results
In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.Conclusions
Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca2+ through a NADPH oxidase-dependent mechanism. 相似文献18.
Background
Of antigen-presenting cells (APCs) expressing HLA-DQ molecules in the celiac disease (CD) lesion, CD11c+ dendritic cells (DCs) co-expressing the monocyte marker CD14 are increased, whereas other DC subsets (CD1c+ or CD103+) and CD163+CD11c− macrophages are all decreased. It is unclear whether these changes result from chronic inflammation or whether they represent early events in the gluten response. We have addressed this in a model of in vivo gluten challenge.Methods
Treated HLA-DQ2+ CD patients (n = 12) and HLA-DQ2+ gluten-sensitive control subjects (n = 12) on a gluten-free diet (GFD) were orally challenged with gluten for three days. Duodenal biopsies obtained before and after gluten challenge were subjected to immunohistochemistry. Single cell digests of duodenal biopsies from healthy controls (n = 4), treated CD (n = 3) and untreated CD (n = 3) patients were analyzed by flow cytometry.Results
In treated CD patients, the gluten challenge increased the density of CD14+CD11c+ DCs, whereas the density of CD103+CD11c+ DCs and CD163+CD11c− macrophages decreased, and the density of CD1c+CD11c+ DCs remained unchanged. Most CD14+CD11c+ DCs co-expressed CCR2. The density of neutrophils also increased in the challenged mucosa, but in most patients no architectural changes or increase of CD3+ intraepithelial lymphocytes (IELs) were found. In control tissue no significant changes were observed.Conclusions
Rapid accumulation of CD14+CD11c+ DCs is specific to CD and precedes changes in mucosal architecture, indicating that this DC subset may be directly involved in the immunopathology of the disease. The expression of CCR2 and CD14 on the accumulating CD11c+ DCs indicates that these cells are newly recruited monocytes. 相似文献19.
Wu CY Jia Z Wang W Ballou LM Jiang YP Chen B Mathias RT Cohen IS Song LS Entcheva E Lin RZ 《PloS one》2011,6(9):e24404
Background
Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.Methods and Results
Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca2+ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca2+ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.Conclusions
PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca2+-induced Ca2+ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials. 相似文献20.