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41.
The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in 1H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)‐indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high‐performance liquid chromatography (HPLC) method based on a modified β‐cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25–1000 μM for the (1R,3S)‐enantiomer and 1.25‐750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)‐enantiomer and up to 99.67% ee for the (1S,3R)‐enantiomer. Chirality 25:923–933, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
42.
David Pretzel Stefanie Linss Hannes Ahrem Michaela Endres Christian Kaps Dieter Klemm Raimund W Kinne 《Arthritis research & therapy》2013,15(3):R59
Introduction
Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.Methods
Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.Results
Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.Conclusions
The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors. 相似文献43.
In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure. 相似文献
44.
Pavlik R Wypior G Hecht S Papadopoulos P Kupka M Thaler C Wiest I Pestka A Friese K Jeschke U 《Histochemistry and cell biology》2011,136(3):289-299
Estradiol and progesterone mediate their actions by binding to classical nuclear receptors, estrogen receptor α (ERα) and
estrogen receptor β (ERβ) and progesterone receptor A and B (PR-A and PR-B) and the non-classical G protein-coupled estrogen
receptor (GPER). Several animal knock-out models have shown the importance of the receptors for growth of the oocyte and ovulation.
The aim of our study was to identify GPER in human granulosa cells (GC) for the first time. Moreover, the effect of different
doses of gonadotropins on estrogen and progesterone receptors in the human ovary should be investigated as follicle stimulating
hormone (FSH) and luteinizing hormone (LH) are also responsible for numerous mechanisms in the ovary like induction of the
steroid biosynthesis. Human GC were cultured in vitro and stimulated with different doses of recombinant human FSH or LH.
Receptor expression was analyzed by immunocytochemistry and quantitative real-time RT-PCR. GPER could be identified for the
first time in human GC. It could be shown that high concentrations of LH increase GPER protein expression. Furthermore FSH
and LH increased ERβ, PR-A and PR-B significantly on protein level. These findings were verified for high doses of FSH and
LH on mRNA level. ERα was not affected with FSH or LH. We assume that gonadotropins induce GPER, ERβ and PR in luteinized
granulosa cells. 相似文献
45.
Toshchakov VY Fenton MJ Vogel SN 《Journal of immunology (Baltimore, Md. : 1950)》2007,178(5):2655-2660
We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-kappaB translocation and early IL-1beta mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys(4)-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-kappaB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins. 相似文献
46.
Soni Pullamsetti Stefanie Krick Hüseyin Yilmaz Hossein Ardeschir Ghofrani Christian Schudt Norbert Weissmann Beate Fuchs Werner Seeger Friedrich Grimminger Ralph Theo Schermuly 《Respiratory research》2005,6(1):128
Background
The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.Methods
CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated.Results
Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 ± 4.0 to 68.9 ± 3.2 after 4 weeks and 74.9 ± 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response.Conclusion
In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics. 相似文献47.
This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H+-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H+ and K+ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H+ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H+ by the hexoseproton symport and charge compensation by K+ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H+-ATPase caused a slow-down of the K+ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H+ uptake and K+ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H+ fluxes. The quantitative participation of the several phases of H+ and K+ flow depended on the pH of the medium, the ambient Ca2+ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H+-ATPase never fully compensated for H+ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K+ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO
dimethyloxazolidinedione
- EDTA
ethylcnediaminetetraacetic acid
- p.c.
packed cell volume 相似文献
48.
Tobias Ruck Stefanie Bock Steffen Pfeuffer Christina B.Schroeter Derya Cengiz Paul Marciniak Maren Lindner Alexander Herrmann Marie Liebmann Stjepana Kovac Lukas Gola Leoni Rolfes Marc Pawlitzki Nils Opel Tim Hahn Udo Dannlowski Thomas Pap Felix Luessi Julian A.Schreiber Bernhard Wünsch Tanja Kuhlmann Guiscard Seebohm Bjrn Tackenberg Patricia Seja Frank Dring Erhard Wischmeyer Achmet Imam Chasan Johannes Roth Luisa Klotz Gerd Meyer zu Hrste Heinz Wiendl Tobias Marschall Stefan Floess Jochen Huehn Thomas Budde Tobias Bopp Stefan Bittner Sven G.Meuth 《Cell research》2022,32(1):72-88
It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv... 相似文献
49.
50.
Katherine E. Horn Stephen D. Glasgow Delphine Gobert Sarah-Jane Bull Tamarah Luk Jacklyn Girgis Marie-Eve Tremblay Danielle McEachern Jean-François Bouchard Michael Haber Edith Hamel Paul Krimpenfort Keith K. Murai Anton Berns Guy Doucet C. Andrew Chapman Edward S. Ruthazer Timothy E. Kennedy 《Cell reports》2013,3(1):173-185
Highlights? DCC and netrin-1 are enriched at synapses in the adult mouse forebrain ? DCC is enriched in the PSD and regulates dendritic spine morphology ? LTP induction and memory formation require DCC expression by neurons ? DCC activation of Src is required for NMDAR-dependent LTP in adult CNS 相似文献