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51.
Li M Alnouti Y Leverence R Bi H Gusev AI 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,825(2):152-160
Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections. 相似文献
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Alexey Stepanov Alexander Belyy Igor Kasheverov Alexandra Rybinets Maria Dronina Igor Dyachenko Arkady Murashev Vera Knorre Dmitry Sakharov Natalya Ponomarenko Victor Tsetlin Alexander Tonevitsky Sergey Deyev Alexey BelogurovJr. Alexander Gabibov 《Biotechnology letters》2016,38(7):1173-1180
Objective
Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.Results
Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT.Conclusion
Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS.53.
54.
Xueping Wu Julia Sagave Arkady Rutkovskiy Fred Haugen Anton Baysa Ståle Nygård Gabor Czibik Christen Peder Dahl Lars Gullestad Jarle Vaage Guro Valen 《Life sciences》2014
Aims
Heart failure is associated with activation of fetal gene programs. Bone morphogenetic proteins (BMPs) regulate embryonic development through interaction with BMP receptors (BMPRs) on the cell surface. We investigated if the expression of BMP4 and its receptors BMPR1a and BMPR2 were activated in post-infarction remodeling and heart failure.Main methods
Left ventricular biopsies were taken from explanted hearts of patients with end-stage heart failure due to dilated cardiomyopathy (CMP; n = 15) or ischemic heart disease (CAD; n = 9), and compared with homograft control preparations from organ donors deceased due to non-cardiac causes (n = 7). Other samples were taken from patients undergoing coronary artery bypass grafting (CABG; n = 11). Mice were subjected to induced infarction by permanent coronary artery ligation or sham operation, and hearts were sampled serially thereafter (n = 7 at each time point).Key findings
Human and mouse hearts expressed BMP4 and both receptor subtypes. CABG and CMP patients had increased expression of mRNA encoding for BMP4, but unchanged protein. Mouse hearts had increased BMP4 precursor protein 24 h after infarction. BMPR1a protein decreased in CAD patients and initially in postinfarcted mouse hearts, but increased again in the latter after two weeks. Human recombinant BMP4 promoted survival after H2O2 injury in HL-1 cells, and also protected adult mouse cardiomyocytes against hypoxia–reoxygenation injury.Significance
Adult hearts express BMP4, the mRNA increasingly so in patients with coronary artery disease with good cardiac function. BMPRs are downregulated in cardiac remodeling and failure. Recombinant BMP4 has protective effects on cultured cardiomyocytes. 相似文献55.
Arkady Mustaev Muhammad Malik Xilin Zhao Natalia Kurepina Gan Luan Lisa M. Oppegard Hiroshi Hiasa Kevin R. Marks Robert J. Kerns James M. Berger Karl Drlica 《The Journal of biological chemistry》2014,289(18):12300-12312
DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly81 and GyrB-Glu466 residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases. 相似文献
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57.
Heng Wu Ying Jin Jaqueline Arias Jorge Bassuk Arkady Uryash Paul Kurlansky Keith Webster Jose A. Adams 《Nitric oxide》2009,20(1):63-68
Periodic acceleration (pGz), sinusoidal motion of the whole body in a head–foot direction in the spinal axis, is a novel noninvasive means for cardiopulmonary support and induction of pulsatile shear stress. pGz increases plasma nitrite levels, in vivo and in vitro. Additionally, pGz confers cardioprotection in models of ischemia reperfusion injury. We hypothesize that pGz may also confer a cardiac phenotypic change by upregulation of the expression of the various NO synthase (NOS) isoforms in vivo. pGz was applied for 1 h to awake restrained male rats at 2 frequencies (360 and 600 cpm) and acceleration (Gz) of ±3.4 m/s2. pGz did not affect arterial blood gases or electrolytes. pGz significantly increased total nitrosylated protein levels, indicating increased NO production. pGz also increased mRNA and protein levels of eNOS and nNOS, and phosphorylated eNOS in heart. pGz increased Akt phosphorylation (p-AKT), but not total Akt, or phosphorylated ERK1/2. Inducible (i) NOS levels were undetectable with or without pGz. Immunoblotting revealed the localization of nNOS, exclusively in cardiomyocyte, and pGz increased its expression. We have demonstrated that pGz changes myocardial NOS phenotypes. Such upregulation of eNOS and nNOS was still evident 24 h after pGz. Further studies are needed to understand the biochemical and biomechanical signal transduction pathway for the observed NOS phenotype changed induced by pGz. 相似文献
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59.
Renal and endocrine changes in rats with inherited stress-induced arterial hypertension (ISIAH) 总被引:2,自引:2,他引:0
Amstislavsky S Welker P Frühauf JH Maslova L Ivanova L Jensen B Markel AL Bachmann S 《Histochemistry and cell biology》2006,125(6):651-659
Hypertensive inbred rats (ISIAH; inherited stress-induced arterial hypertension) present with baseline hypertension (>170 mmHg in adult rats), but attain substantially higher values upon mild emotional stress. We aimed to characterize key parameters related to hypertension in ISIAH. Kidneys, adrenals, and systemic endocrine parameters were studied in ISIAH of different ages and compared to normotensive Wistar albino Glaxo (WAG) rats. Native organs were obtained for Western and PCR analysis. Perfusion-fixed organs were prepared for histopathology and quantitative histochemistry. Plasma renin and adrenal hormones were measured. Renal morphology was unaltered in ISIAH. The hypothalamic-pituitary-adrenocortical (HPA) axis was constitutively upregulated with enlarged adrenal cortices and enhanced plasma corticosterone levels. Plasma renin activity was not different between groups, whereas aldosterone levels were in part reduced. Juxtaglomerular NO synthase type 1, cyclooxygenase type 2, and renin expression were significantly reduced, whereas tubular gene products related to sodium transport (bumetanide-sensitive Na, K, 2Cl cotransporter type 2; thiazide-sensitive Na, Cl cotransporter; epithelial Na channel-α; 11β-hydroxysteroid dehydrogenase type 2) were increased. These data suggest enhanced volume conservation by the kidney. Our data define ISIAH as an attractive model for the renal components determining salt and water homeostasis in hypertension. The specific condition of a basally stimulated HPA axis is highlighted, including the option to study effects superimposed by emotional stress. 相似文献
60.
A Rutkovskiy LH Mariero S Nygård KO Stensløkken G Valen J Vaage 《Biochemical and biophysical research communications》2012,421(1):70-75
Angiotensin-I converting enzyme (ACE) is a zinc dependent peptidase with a major role in regulating vasoactive peptide metabolism. ACE, a transmembrane protein, undergoes proteolysis, or shedding, by an as yet unidentified proteinase to release a catalytically active soluble form of the enzyme. Physiologically, soluble ACE in plasma is derived primarily from endothelial cells. We demonstrate that ACE shedding from confluent endothelial cells is increased in response to bacterial lipopolysaccharide, but not phorbol esters. Characterisation of lipopolysaccharide stimulated shedding showed that there is a lag phase before soluble ACE can be detected which is sensitive to inhibitors of translation, NF-κB, TNFα and TNFR-I/II. The shedding phase is less sensitive to these inhibitors, but is ablated by BB-94, a Matrix Metalloproteinase (MMP)/A Disintegrin and Metalloproteinase (ADAM) inhibitor. Tissue Inhibitor of Metalloproteinase (TIMP) profiling suggested a requirement for ADAM9 in lipopolysaccharide induced ACE shedding, which was confirmed by depletion with siRNA. Transient transfection of ADAM9 and ACE cDNAs into HEK293 cells demonstrated that ADAM9 requires both membrane anchorage and its catalytic domain to shed ACE. 相似文献