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1.
Hervé Boutin Christian Prenant Renaud Maroy James Galea Andrew D. Greenhalgh Alison Smigova Christopher Cawthorne Peter Julyan Shane M. Wilkinson Samuel D. Banister Gavin Brown Karl Herholz Michael Kassiou Nancy J. Rothwell 《PloS one》2013,8(2)
Purpose
Neuroinflammation is involved in several brain disorders and can be monitored through expression of the translocator protein 18 kDa (TSPO) on activated microglia. In recent years, several new PET radioligands for TSPO have been evaluated in disease models. [18F]DPA-714 is a TSPO radiotracer with great promise; however results vary between different experimental models of neuroinflammation. To further examine the potential of [18F]DPA-714, it was compared directly to [11C]PK11195 in experimental cerebral ischaemia in rats.Methods
Under anaesthesia, the middle cerebral artery of adult rats was occluded for 60 min using the filament model. Rats were allowed recovery for 5 to 7 days before one hour dynamic PET scans with [11C]PK11195 and/or [18F]DPA-714 under anaesthesia.Results
Uptake of [11C]PK11195 vs [18F]DPA-714 in the ischemic lesion was similar (core/contralateral ratio: 2.84±0.67 vs 2.28±0.34 respectively), but severity of the brain ischemia and hence ligand uptake in the lesion appeared to vary greatly between animals scanned with [11C]PK11195 or with [18F]DPA-714. To solve this issue of inter-individual variability, we performed a direct comparison of [11C]PK11195 and [18F]DPA-714 by scanning the same animals sequentially with both tracers within 24 h. In this direct comparison, the core/contralateral ratio (3.35±1.21 vs 4.66±2.50 for [11C]PK11195 vs [18F]DPA-714 respectively) showed a significantly better signal-to-noise ratio (1.6 (1.3–1.9, 95%CI) fold by linear regression) for [18F]DPA-714.Conclusions
In a clinically relevant model of neuroinflammation, uptake for both radiotracers appeared to be similar at first, but a high variability was observed in our model. Therefore, to truly compare tracers in such models, we performed scans with both tracers in the same animals. By doing so, our result demonstrated that [18F]DPA-714 displayed a higher signal-to-noise ratio than [11C]PK11195. Our results suggest that, with the longer half-life of [18F] which facilitates distribution of the tracer across PET centre, [18F]DPA-714 is a good alternative for TSPO imaging. 相似文献2.
Paolo Zanotti-Fregonara Jussi Hirvonen Chul Hyoung Lyoo Sami S. Zoghbi Denise Rallis-Frutos Marilyn A. Huestis Cheryl Morse Victor W. Pike Robert B. Innis 《PloS one》2013,8(4)
Background
Population-based input function (PBIF) may be a valid alternative to full blood sampling for quantitative PET imaging. PBIF is typically validated by comparing its quantification results with those obtained via arterial sampling. However, for PBIF to be employed in actual clinical research studies, its ability to faithfully capture the whole spectrum of results must be assessed. The present study validated a PBIF for [18F]FMPEP-d 2, a cannabinoid CB1 receptor radioligand, in healthy volunteers, and also attempted to utilize PBIF to replicate three previously published clinical studies in which the input function was acquired with arterial sampling.Methods
The PBIF was first created and validated with data from 42 healthy volunteers. This PBIF was used to assess the retest variability of [18F]FMPEP-d 2, and then to quantify CB1 receptors in alcoholic patients (n = 18) and chronic daily cannabis smokers (n = 29). Both groups were scanned at baseline and after 2–4 weeks of monitored drug abstinence.Results
PBIF yielded accurate results in the 42 healthy subjects (average Logan-distribution volume (V T) was 13.3±3.8 mL/cm3 for full sampling and 13.2±3.8 mL/cm3 for PBIF; R2 = 0.8765, p<0.0001) and test-retest results were comparable to those obtained with full sampling (variability: 16%; intraclass correlation coefficient: 0.89). PBIF accurately replicated the alcoholism study, showing a widespread ∼20% reduction of CB1 receptors in alcoholic subjects, without significant change after abstinence. However, a small PBIF-V T bias of −9% was unexpectedly observed in cannabis smokers. This bias led to substantial errors, including a V T decrease in regions that had shown no downregulation in the full input function. Simulated data showed that the original findings could only have been replicated with a PBIF bias between −6% and +4%.Conclusions
Despite being initially well validated in healthy subjects, PBIF may misrepresent clinical protocol results and be a source of variability between different studies and institutions. 相似文献3.
Shigeyuki Yamamoto Yasuomi Ouchi Daisaku Nakatsuka Tsuyoshi Tahara Kei Mizuno Seiki Tajima Hirotaka Onoe Etsuji Yoshikawa Hideo Tsukada Masao Iwase Kouzi Yamaguti Hirohiko Kuratsune Yasuyoshi Watanabe 《PloS one》2012,7(12)
Background
Numerous associations between brain-reactive antibodies and neurological or psychiatric symptoms have been proposed. Serum autoantibody against the muscarinic cholinergic receptor (mAChR) was increased in some patients with chronic fatigue syndrome (CFS) or psychiatric disease. We examined whether serum autoantibody against mAChR affected the central cholinergic system by measuring brain mAChR binding and acetylcholinesterase activity using positron emission tomography (PET) in CFS patients with positive [CFS(+)] and negative [CFS(−)] autoantibodies.Methodology
Five CFS(+) and six CFS(−) patients, as well as 11 normal control subjects underwent a series of PET measurements with N-[11C]methyl-3-piperidyl benzilate [11C](+)3-MPB for the mAChR binding and N-[11C]methyl-4-piperidyl acetate [11C]MP4A for acetylcholinesterase activity. Cognitive function of all subjects was assessed by neuropsychological tests. Although the brain [11C](+)3-MPB binding in CFS(−) patients did not differ from normal controls, CFS(+) patients showed significantly lower [11C](+)3-MPB binding than CFS(−) patients and normal controls. In contrast, the [11C]MP4A index showed no significant differences among these three groups. Neuropsychological measures were similar among groups.Conclusion
The present results demonstrate that serum autoantibody against the mAChR can affect the brain mAChR without altering acetylcholinesterase activity and cognitive functions in CFS patients. 相似文献4.
Q Cheng L Lu J Grafström MH Olofsson JO Thorell E Samén K Johansson HS Ahlzén S Stone-Elander S Linder ES Arnér;Sel-tag Imaging Project 《PloS one》2012,7(8):e42151
Background
In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model.Methodology/Principal Findings
Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on 11C-labeled Sel-tagged Annexin A5 ([11C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [11C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [18F]-FDG uptake was not affected by this treatment in this tumor model. [11C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session.Conclusions/Significance
The combined use of dynamic PET with [11C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors. 相似文献5.
Luping Zhou Olivier Salvado Vincent Dore Pierrick Bourgeat Parnesh Raniga S. Lance Macaulay David Ames Colin L. Masters Kathryn A. Ellis Victor L. Villemagne Christopher C. Rowe Jurgen Fripp AIBL Research Group 《PloS one》2014,9(1)
Background
β-amyloid (Aβ) plaques in brain''s grey matter (GM) are one of the pathological hallmarks of Alzheimer''s disease (AD), and can be imaged in vivo using Positron Emission Tomography (PET) with 11C or 18F radiotracers. Estimating Aβ burden in cortical GM has been shown to improve diagnosis and monitoring of AD. However, lacking structural information in PET images requires such assessments to be performed with anatomical MRI scans, which may not be available at different clinical settings or being contraindicated for particular reasons. This study aimed to develop an MR-less Aβ imaging quantification method that requires only PET images for reliable Aβ burden estimations.Materials and Methods
The proposed method has been developed using a multi-atlas based approach on 11C-PiB scans from 143 subjects (75 PiB+ and 68 PiB- subjects) in AIBL study. A subset of 20 subjects (PET and MRI) were used as atlases: 1) MRI images were co-registered with tissue segmentation; 2) 3D surface at the GM-WM interfacing was extracted and registered to a canonical space; 3) Mean PiB retention within GM was estimated and mapped to the surface. For other participants, each atlas PET image (and surface) was registered to the subject''s PET image for PiB estimation within GM. The results are combined by subject-specific atlas selection and Bayesian fusion to generate estimated surface values.Results
All PiB+ subjects (N = 75) were highly correlated between the MR-dependent and the PET-only methods with Intraclass Correlation (ICC) of 0.94, and an average relative difference error of 13% (or 0.23 SUVR) per surface vertex. All PiB- subjects (N = 68) revealed visually akin patterns with a relative difference error of 16% (or 0.19 SUVR) per surface vertex.Conclusion
The demonstrated accuracy suggests that the proposed method could be an effective clinical inspection tool for Aβ imaging scans when MRI images are unavailable. 相似文献6.
María Luisa Soto-Montenegro Santiago Pe?a-Zalbidea Jose María Mateos-Pérez Marta Oteo Eduardo Romero Miguel ángel Morcillo Manuel Desco 《PloS one》2014,9(11)
Purpose
The goal of this study was to compare the tumor uptake kinetics and diagnostic value of three 68Ga-DOTA-labeled somatostatin analogues (68Ga-DOTATOC, 68Ga-DOTANOC, and 68Ga-DOTATATE) using PET/CT in a murine model with subcutaneous meningioma xenografts.Methods
The experiment was performed with 16 male NUDE NU/NU mice bearing xenografts of a human meningioma cell line (CH-157MN). 68Ga-DOTATOC, 68Ga-DOTANOC, and 68Ga-DOTATATE were produced in a FASTLab automated platform. Imaging was performed on an Argus small-animal PET/CT scanner. The SUVmax of the liver and muscle, and the tumor-to-liver (T/L) and tumor-to-muscle (T/M) SUV ratios were computed. Kinetic analysis was performed using Logan graphical analysis for a two-tissue reversible compartmental model, and the volume of distribution (Vt) was determined.Results
Hepatic SUVmax and Vt were significantly higher with 68Ga-DOTANOC than with 68Ga-DOTATOC and 68Ga-DOTATATE. No significant differences between tracers were found for SUVmax in tumor or muscle. No differences were found in the T/L SUV ratio between 68Ga-DOTATATE and 68Ga-DOTATOC, both of which had a higher fraction than 68Ga-DOTANOC. The T/M SUV ratio was significantly higher with 68Ga-DOTATATE than with 68Ga-DOTATOC and 68Ga-DOTANOC. The Vt for tumor was higher with 68Ga-DOTATATE than with 68Ga-DOTANOC and relatively similar to that of 68Ga-DOTATOC.Conclusions
This study demonstrates, for the first time, the ability of the three radiolabeled somatostatin analogues tested to image a human meningioma cell line. Although Vt was relatively similar with 68Ga-DOTATATE and 68Ga-DOTATOC, uptake was higher with 68Ga-DOTATATE in the tumor than with 68Ga-DOTANOC and 68Ga-DOTATOC, suggesting a higher diagnostic value of 68Ga-DOTATATE for detecting meningiomas. 相似文献7.
Background and Aims
The main assemblage of the grass subfamily Chloridoideae is the largest known clade of C4 plant species, with the notable exception of Eragrostis walteri Pilg., whose leaf anatomy has been described as typical of C3 plants. Eragrostis walteri is therefore classically hypothesized to represent an exceptional example of evolutionary reversion from C4 to C3 photosynthesis. Here this hypothesis is tested by verifying the photosynthetic type of E. walteri and its classification.Methods
Carbon isotope analyses were used to determine the photosynthetic pathway of several E. walteri accessions, and phylogenetic analyses of plastid rbcL and ndhF and nuclear internal transcribed spacer DNA sequences were used to establish the phylogenetic position of the species.Results
Carbon isotope analyses confirmed that E. walteri is a C3 plant. However, phylogenetic analyses demonstrate that this species has been misclassified, showing that E. walteri is positioned outside Chloridoideae in Arundinoideae, a subfamily comprised entirely of C3 species.Conclusions
The long-standing hypothesis of C4 to C3 reversion in E. walteri is rejected, and the classification of this species needs to be re-evaluated. 相似文献8.
Purpose
To develop a tri-modality image fusion method for better target delineation in image-guided radiotherapy for patients with brain tumors.Methods
A new method of tri-modality image fusion was developed, which can fuse and display all image sets in one panel and one operation. And a feasibility study in gross tumor volume (GTV) delineation using data from three patients with brain tumors was conducted, which included images of simulation CT, MRI, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) examinations before radiotherapy. Tri-modality image fusion was implemented after image registrations of CT+PET and CT+MRI, and the transparency weight of each modality could be adjusted and set by users. Three radiation oncologists delineated GTVs for all patients using dual-modality (MRI/CT) and tri-modality (MRI/CT/PET) image fusion respectively. Inter-observer variation was assessed by the coefficient of variation (COV), the average distance between surface and centroid (ADSC), and the local standard deviation (SDlocal). Analysis of COV was also performed to evaluate intra-observer volume variation.Results
The inter-observer variation analysis showed that, the mean COV was 0.14(±0.09) and 0.07(±0.01) for dual-modality and tri-modality respectively; the standard deviation of ADSC was significantly reduced (p<0.05) with tri-modality; SDlocal averaged over median GTV surface was reduced in patient 2 (from 0.57 cm to 0.39 cm) and patient 3 (from 0.42 cm to 0.36 cm) with the new method. The intra-observer volume variation was also significantly reduced (p = 0.00) with the tri-modality method as compared with using the dual-modality method.Conclusion
With the new tri-modality image fusion method smaller inter- and intra-observer variation in GTV definition for the brain tumors can be achieved, which improves the consistency and accuracy for target delineation in individualized radiotherapy. 相似文献9.
Agustina Mariana Portu Andrés Eugenio Rossini Mario Alberto Gadan Omar Alberto Bernaola Silvia Inés Thorp Paula Curotto Emiliano César Cayetano Pozzi Rómulo Luis Cabrini Gisela Saint Martin 《Reports of Practical Oncology and Radiotherapy》2016,21(2):129-134
Aim
In this work we present a methodology to produce an “imprint” of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation.Background
The distribution and concentration of 10B atoms in tissue samples coming from BNCT (Boron Neutron Capture Therapy) protocols can be determined through the quantification and analysis of the tracks forming its autoradiography image on a nuclear track detector. The location of boron atoms in the cell structure could be known more accurately by the simultaneous observation of the nuclear tracks and the sample image on the detector.Materials and Methods
A UV C irradiator was constructed. The irradiance was measured along the lamp direction and at different distances. Melanoma cells were cultured on polycarbonate foils, incubated with borophenylalanine, irradiated with thermal neutrons and exposed to UV C radiation. The samples were chemically attacked with a KOH solution.Results
A uniform irradiation field was established to expose the detector foils to UV C light. Cells could be seeded on the polycarbonate surface. Both imprints from cells and nuclear tracks were obtained after chemical etching.Conclusions
It is possible to yield cellular imprints in polycarbonate. The nuclear tracks were mostly present inside the cells, indicating a preferential boron uptake. 相似文献10.
Background
Globally plants are the primary sink of atmospheric CO2, but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important.Methodology
We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either 13CO2 to leaves or 13C-glucose to shoots via xylem uptake. The translocation of 13CO2 from the source to other plant parts could be traced by 13C-labeled isoprene and respiratory 13CO2 emission.Principal Finding
In intact plants, assimilated 13CO2 was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h−1. 13C label was stored in the roots and partially reallocated to the plants'' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76–78%) from recently fixed CO2, to a minor extent from xylem-transported sugars (7–11%) and from photosynthetic intermediates with slower turnover rates (8–11%).Conclusion
We quantified the plants'' C loss as respiratory CO2 and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux. 相似文献11.
Ming-Lun Yeh Chung-Feng Huang Ching-I Huang Shu-Fen Liu Hua-Ling Yang Ming-Yen Hsieh Jee-Fu Huang Chia-Yen Dai Wan-Long Chuang Ming-Lung Yu 《PloS one》2014,9(7)
Background
Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV.Objectives
This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan).Study Design
One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT).Results
The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R2 = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log10 IU/ml was not as good as in all samples (R2 = 0.457). Overall, 21.5% of samples had a detection difference of ≥1 log10 IU/ml with 91.9% of these having HBV DNA level <3 log10 IU/ml. Twenty-four patients experiencedVBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime.Conclusions
RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation. 相似文献12.
Jobin Mathew Savitha Balakrishnan Sherin Antony Pretty Mary Abraham CS Paulose 《Journal of biomedical science》2012,19(1):25
Abstact
Background
Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue.Methods
In the present study, alterations of the general GABA, GABAA and GABAB receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated.Results
Scatchard analysis of [3H]GABA, [3H]bicuculline and [3H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in Bmax (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABAAά1, GABAAγ, GABAAδ, GABAB and GAD where down regulated (P < 0.001) in epileptic rats. GABAAά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance.Conclusions
Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management. 相似文献13.
Cheung CY Tso AW Cheung BM Xu A Fong CH Ong KL Law LS Wat NM Janus ED Sham PC Lam KS 《PloS one》2011,6(12):e28598
Context
The KCNJ11 E23K variant is associated with type 2 diabetes mellitus (T2DM) in cross-sectional studies, but conflicting findings have been reported from prospective studies.Objective
This study aimed to evaluate whether the E23K variant could predict glycaemic progression in a Southern Chinese population.Methods/Principal Findings
We performed a long-term prospective study on 1912 subjects from the Hong Kong Cardiovascular Risk Factors Prevalence Study (CRISPS). The KCNJ11 E23K variant was associated with the progression to prediabetes after a median interval of 12 years on multinomial logistic regression analysis, even after adjustment for traditional risk factors (OR 1.29, Page, sex, BMI and fasting plasma glucose [FPG] adjusted = 0.02). Based on Cox proportional hazard regression analysis, the E23K variant also predicted incident prediabetes (HR 1.18, Page, sex, BMI and FPG adjusted = 0.021). However, E23K was not associated with the progression to T2DM in either multinomial or Cox regression analysis, and the association of E23K with glycaemic progression to either prediabetes or T2DM was significant only in unadjusted Cox regression analysis (P = 0.039). In a meta-analysis of eight prospective studies including our own, involving 15680 subjects, the E23K variant was associated with incident T2DM (fixed effect: OR 1.10, P = 4×10−3; random effect: OR 1.11, P = 0.035).Conclusions
Our study has provided supporting evidence for the role of the E23K variant in glycaemic progression in Chinese, with its effect being more evident in the early stage of T2DM, as the subjects progressed from normal glucose tolerance to prediabetes. 相似文献14.
Isik Turker Chih-Chieh Yu Po-Cheng Chang Zhenhui Chen Yoshiro Sohma Shien-Fong Lin Peng-Sheng Chen Tomohiko Ai 《PloS one》2013,8(7)
Background
Apamin sensitive potassium current (I KAS), carried by the type 2 small conductance Ca2+-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.Objective
To test the hypothesis that amiodarone inhibits I KAS in human embryonic kidney 293 (HEK-293) cells.Methods
We used the patch-clamp technique to study I KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.Results
Amiodarone inhibited IKAS in a dose-dependent manner (IC50, 2.67±0.25 µM with 1 µM intrapipette Ca2+). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of IKAS induced with 1 µM intrapipette Ca2+ (n = 3). IKAS inhibition by amiodarone was not voltage-dependent, but was Ca2+-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I KAS induced with 1 µM Ca2+ (n = 4), and 16.4±4.9% with 250 nM Ca2+ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca2+ dependent inhibition of I KAS.Conclusion
Both amiodarone and desethylamiodarone inhibit I KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca2+ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles. 相似文献15.
Background
Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies.Principal Findings
We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1).Significance
The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required. 相似文献16.
Introduction
Glucocorticoids are commonly used as therapeutic agents in many acute and chronic inflammatory and auto-immune diseases. The current study investigated the effects of methylprednisolone (a synthetic glucocorticoid) on aortic distensibility and vascular resistance in lipopolysaccharide-induced chronic inflammation in male Wistar rats.Methods
Chronic inflammation was induced by implanting a subcutaneous slow-release ALZET osmotic pump (1 mg kg−1 day−1 lipopolysaccharide) for either 2 or 4 weeks. Arterial wave transit time (τ) was derived to describe the elastic properties of aortas using the impulse response function of the filtered aortic input impedance spectra.Results
Long-term lipopolysaccharide challenge enhanced the expression of advanced glycation end products (AGEs) in the aortas. Lipopolysaccharide also upregulated the inducible form of nitric oxide synthase to produce high levels of nitric oxide (NO), which resulted in vasodilation, as evidenced by the fall in total peripheral resistance (Rp). However, lipopolysaccharide challenge did not influence the elastic properties of aortas, as shown by the unaltered τ. The NO-mediated vascular relaxation may counterbalance the AGEs-induced arterial stiffening so that the aortic distensibility remained unaltered. Treating lipopolysaccharide-challenged rats with methylprednisolone prevented peripheral vasodilation because of its ability to increase Rp. However, methylprednisolone produced an increase in aorta stiffness, as manifested by the significant decline in τ. The diminished aortic distensibility by methylprednisolone paralleled a significant reduction in NO plasma levels, in the absence of any significant changes in AGEs content.Conclusion
Methylprednisolone stiffens aortas and elastic arteries in lipopolysaccharide-induced chronic inflammation in rats, for NO activity may be dominant as a counteraction of AGEs. 相似文献17.
Background and Aims
The success of C4 plants lies in their ability to attain greater efficiencies of light, water and nitrogen use under high temperature, providing an advantage in arid, hot environments. However, C4 grasses are not necessarily less sensitive to drought than C3 grasses and are proposed to respond with greater metabolic limitations, while the C3 response is predominantly stomatal. The aims of this study were to compare the drought and recovery responses of co-occurring C3 and C4 NADP-ME grasses from the subfamily Panicoideae and to determine stomatal and metabolic contributions to the observed response.Methods
Six species of locally co-occurring grasses, C3 species Alloteropsis semialata subsp. eckloniana, Panicum aequinerve and Panicum ecklonii, and C4 (NADP-ME) species Heteropogon contortus, Themeda triandra and Tristachya leucothrix, were established in pots then subjected to a controlled drought followed by re-watering. Water potentials, leaf gas exchange and the response of photosynthetic rate to internal CO2 concentrations were determined on selected occasions during the drought and re-watering treatments and compared between species and photosynthetic types.Key Results
Leaves of C4 species of grasses maintained their photosynthetic advantage until water deficits became severe, but lost their water-use advantage even under conditions of mild drought. Declining C4 photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO2 assimilation, whereas, in the C3 species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C4 plants could explain the observed slower recovery of photosynthesis on re-watering, in comparison with C3 plants which recovered a greater proportion of photosynthesis through increased stomatal conductance.Conclusions
Within the Panicoid grasses, C4 (NADP-ME) species are metabolically more sensitive to drought than C3 species and recover more slowly from drought. 相似文献18.
Lucy Vivash Marie-Claude Gregoire Viviane Bouilleret Alexis Berard Catriona Wimberley David Binns Peter Roselt Andrew Katsifis Damian E. Myers Rodney J. Hicks Terence J. O'Brien Stefanie Dedeurwaerdere 《PloS one》2014,9(1)
Purpose
Imbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether [18F]-flumazenil ([18F]-FMZ) PET could be used to non-invasively characterise GABAA/central benzodiazepine receptor (GABAA/cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE.Methods
Dynamic [18F]-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5–25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an [18F]-FMZ PET scan (3.6–4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the [18F]-FMZ PET data.Key Findings
The PSM was found to adequately model [18F]-FMZ binding in vivo. There was a significant decrease in hippocampal receptor density in the SE group (p<0.01), accompanied by an increase in apparent affinity (p<0.05) compared to controls. No change in cortical receptor binding was observed. Hippocampal volume reduction and cell loss was only seen in a subset of animals. Histological assessment of hippocampal cell loss was significantly correlated with hippocampal volume measured by MRI (p<0.05), but did not correlate with [18F]-FMZ binding.Significance
Alterations to hippocampal GABAA/cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with [18F]-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. [18F]-FMZ PET is useful for investigating the role that changes GABAA/cBZR density and binding affinity play in the pathogenesis of TLE. 相似文献19.
Background
Aldosterone synthase (CYP11B2) T-344C gene polymorphism was found to be correlated with atrial fibrillation (AF) risk. However, the results of individual studies remain conflicting.Objective and methods
A meta-analysis including 2,758 subjects from six individual studies was performed to explore the correlation between CYP11B2 T-344C gene polymorphisms and AF. The pooled odds ratios (ORs) and their corresponding 95% confidence intervals (95% CIs) were evaluated by the fixed– or random–effects model.Results
A significant relationship between CYP11B2 T-344C gene polymorphism and AF was found under allelic (OR: 1.26, 95% CI: 1.11–1.42, P = 0.0002), recessive (OR: 1.99, 95% CI: 1.26–3.14, P = 0.003), dominant (OR: 0.903, 95% CI: 0.820–0.994, P = 0.036), homozygous (OR: 1.356, 95% CI: 1.130–1.628, P = 0.001), and additive (OR: 1.153, 95% CI: 1.070–1.243, P = 1.0×10−10) genetic models. No significant association between CYP11B2 T-344C gene polymorphism and AF was found under the heterozygous genetic model (OR: 1.040, 95% CI: 0.956–1.131, P = 0.361).Conclusions
A significant association was found between CYP11B2 T-344C gene polymorphism and AF risk. Individuals with the C allele of CYP11B2 T-344C gene polymorphism have higher risk for AF. 相似文献20.