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121.
A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise. 相似文献
122.
Richter W Hermsdorf T Lilie H Egerland U Rudolph R Kronbach T Dettmer D 《Protein expression and purification》2000,19(3):375-383
A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates. 相似文献
123.
We explored the use of the reversible cross-linking reagent dimethyl 3,3-dithiobispropionimidate (DTBP) in combination with CO treatment as an approach to stabilizing erythrocyte structure and function. Erythrocytes were cross-linked with different concentrations of DTBP for different times. DTBP increased erythrocyte osmotic stability, blocked lysolecithin-induced echinocytosis, and decreased erythrocyte deformability in a concentration- and time-dependent manner. Reversal of the cross-linking with the reducing agent dithioerythritol (DTE) restored osmotic fragility and response to lysolecithin as well as deformability. Complete reversal, however, is a function of the DTBP concentration and the time of cross-linking. The effects of cross-linking with 5 mM DTBP for 1 h were completely reversible after treatment with 10 mM DTE for 20 min. Longer incubation times or higher concentrations of DTBP resulted in partial reversal by DTE of the effects produced by DTBP. Cross-linking and reversal only slightly reduced the ATP content. The hemoglobin contained in the cross-linked and reversed cells could still undergo reversible oxygenation and deoxygenation. Erythrocytes were pretreated with CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded with a solution containing 500 mM glucose for 24 h, and freeze-dried in a medium containing 15% (w/v) albumin. Rehydration followed in distilled water. Complete recovery, measured as the percentage of free hemoglobin, was achieved for cells cross-linked with 5 mM DTBP for 3 h and freeze-dried to a final water content of 10-15%. Non-cross-linked cells lysed 100% on rehydration in distilled water. No methemoglobin (MetHb) formation as a result of freeze-drying was detected in CO-treated cells. In non-CO-treated cells 20% of the Hb was converted to MetHb. 相似文献
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125.
Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1
P. C. Trackman Rudolph J. Graham Howard K. Bittner David L. Carnes James A. Gilles Dana T. Graves 《Histochemistry and cell biology》1998,110(1):9-14
Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors,
and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously
condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix.
In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory
rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed
oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of
administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions
on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective
tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete
distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells
with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of
a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new
in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression
in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the
regulation of extracellular matrix accumulation.
Accepted: 19 December 1998 相似文献
126.
The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation
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Gabriele Dürr Jochen Strayle Richard Plemper Saskia Elbs Saskia K. Klee Patrice Catty Dieter H. Wolf Hans K. Rudolph 《Molecular biology of the cell》1998,9(5):1149-1162
The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes. 相似文献
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129.
April M. Weissmiller Orlangie Natera-Naranjo Sol M. Reyna Matthew L. Pearn Xiaobei Zhao Phuong Nguyen Soan Cheng Lawrence S. B. Goldstein Rudolph E. Tanzi Steven L. Wagner William C. Mobley Chengbiao Wu 《PloS one》2015,10(2)
Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aβ peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aβ peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aβ levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative. 相似文献