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排序方式: 共有526条查询结果,搜索用时 171 毫秒
111.
Henrik B. Mortensen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1980,182(3):325-333
A method for the quantitation of hemoglobin A1c using isoelectric focusing is reported.Hemolysates were prepared and stabilised with carbon monoxide and with potassium cyanide before the quantitation. The two preparations gave identical results. The potassium cyanide method is simple and adequate for routine purposes, but the cyanohemiglobin compound remains stable for one week only. The carbon monoxide method is more laborious, but the carboxyhemoglobin derivative remains stable for up to one year.Quantitations of the separated fractions emerging after isoelectric focusing were made with spectrophotometry or with densitometry. No significant difference in the results could be shown. Reproducibility tests were improved by introducing transferrin as an internal standard.The specificity of the method was checked by the in vitro addition of oral hypoglycemic drugs and of insulin. 相似文献
112.
N. J. McC. Mortensen Dr. J. F. Morris M.D. C. J. Owens 《Cell and tissue research》1978,192(3):513-525
The effect of intragastric administration of acetylcholine on serum and antral gastrin concentrations of rats has been examined using a radioimmunoassay and quantitative electron microscopy. Exposure of the stomach of rats, previously fasted for 24h, to 2% acetylcholine for either 0.5 or 2h resulted in a significant 4--5 fold increase in serum gastrin concentrations to levels similar to those found in fed animals. Such treatment produced no detectable change in antral gastrin concentration or in the number or electron density of secretory granules in the G cells. This lack of detectable change in the G cells was not unexpected since our calculations suggest that less than 10% of the total gastrin stored in the antrum is released over 2h as a result of the stimulation with acetylcholine. The proportion of electron-lucent secretory granules was, however, markedly increased by prolonged fixation in aldehydes. The increase was similar in both ACh stimulated and control animals. These results indicate that the ultrastructural appearance of G cell secretory granules in influenced far more by the conditions of fixation than by the release of gastrin. They therefore cast considerable doubt on the hypothesis that gastrin is released by molecular dispersion from the granules. 相似文献
113.
Steen Hansen Rogert Bauer Stig Bredsted Lomholt Karsten Bruun Quist Jan Skov Pedersen Kell Mortensen 《European biophysics journal : EBJ》1996,24(3):143-147
The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100–120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40–50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy.
Correspondence to: S. Hansen 相似文献
114.
115.
116.
Feng Q Düring L de Mayolo AA Lettier G Lisby M Erdeniz N Mortensen UH Rothstein R 《DNA Repair》2007,6(1):27-37
Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination. 相似文献
117.
Gori K Hébraud M Chambon C Mortensen HD Arneborg N Jespersen L 《FEMS yeast research》2007,7(2):293-303
The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed. 相似文献
118.
The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use 总被引:1,自引:0,他引:1
BACKGROUND: Studies have shown that stem cell therapy could be a novel option for improving neovascularization and cardiac function in patients with ischemic heart disease. Human mesenchymal stromal cells (MSC) have generated wide interest in the clinical setting because of their ability to regenerate tissue. The aim of the study was to test whether freezing and storage of human BM mononuclear cells (BM-MNC) and ex vivo-expanded MSC influenced their phenotypic and functional characteristics as well as proliferation capacity. METHODS: MNC were isolated from BM and divided into two portions: one part was immediately cultured (MSC P0) whereas the second part was frozen for a week before cultivation and analysis (F-MSC P1). Confluent MSC (P0) were harvested and divided: one was analyzed as MSC P1 and the other was frozen for a week before further cultivation and analysis as F-MSC P2. RESULTS: MSC P1, F-MSC P1 and F-MSC P2 had similar proliferation capacities and demonstrated almost identical expression levels of markers characteristic for MSC. The capacity to form endothelial vascular structures was independent of freezing. DISCUSSION: The proliferation and differentiation capacity as well as the cellular characteristics were identical in cultivated MSC derived from freshly isolated BM-MNC and MSC derived after freezing and storage of either freshly isolated BM-MNC or ex vivo-cultivated MSC. This highlights the potential clinical use of MSC in patients with cardiac and degenerative diseases, as it would be possible to inject MSC obtained from the same BM aspiration at different time points. 相似文献
119.
Sonja Aits Jennifer Kricker Bin Liu Anne-Marie Ellegaard Saara H?m?list? Siri Tvingsholm Elisabeth Corcelle-Termeau S?ren H?gh Thomas Farkas Anna Holm Jonassen Irina Gromova Monika Mortensen Marja J??ttel? 《Autophagy》2015,11(8):1408-1424
Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation. 相似文献
120.
Grant E. Blouse Kenneth A. B?tkj?r Elena Deryugina Aleksandra A. Byszuk Janni M. Jensen Kim K. Mortensen James P. Quigley Peter A. Andreasen 《The Journal of biological chemistry》2009,284(7):4647-4657
Serine proteases are secreted from cells as single-chain zymogens,
typically having activities orders of magnitude lower than those of the mature
two-chain enzymes. Activation occurs by a conformational change initiated by
cleavage of a specific peptide bond. We have derived a monoclonal antibody
(mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form
of urokinase-type plasminogen activator (uPA). We mapped the epitope of the
antibody to the autolysis loop, one of the structural elements known to change
conformation during zymogen activation. A mechanistic evaluation with
biophysical methods elucidated a novel bifunctional inhibitory mechanism
whereby mAb-112 not only delays the proteolytic conversion of single-chain
pro-uPA into the two-chain form but also subsequently averts the
conformational transition to a mature protease by sequestering the two-chain
form in a zymogen-like, noncatalytic state. Functional studies employing two
variants of human HT-1080 cells, exhibiting high and low levels of
dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is
an effective inhibitor of tumor cell intravasation. These findings show that
pharmacological interference with zymogen activation is a plausible and robust
means to regulate uPA activity and the downstream effects of plasminogen
activation in the spread of cancer and other processes of pathological tissue
remodeling. A strategy that targets regions related to pro-enzyme activation
likely provide a unique inhibitor-protease interaction surface and is, thus,
expected to enhance the chances of engineering high inhibitor specificity. Our
results provide new information about the structural flexibility underlying
the equilibrium between active and inactive forms of serine proteases.In nature a key mechanism for regulation of serine proteases with a
trypsin-like fold is the activation of secreted zymogens or proenzymes, which
typically have activities orders of magnitude lower than the mature enzymes.
Zymogen activation is the central step in natural protease cascade regulation,
allowing for rapid amplification of the activation signal. The catalytic
activity of a zymogen relative to the mature protease can generally be thought
of as a problem of equilibrium between active and inactive conformational
states of the protease domain. Zymogen activation generally occurs by cleavage
of the bond between amino acid residues 15 and
16.2 The liberated
amino terminus inserts into a hydrophobic binding cleft of the catalytic
domain forming, in addition to hydrophobic interactions, a salt bridge to the
side chain of Asp194 which stabilizes the substrate binding pocket
and oxyanion hole in a catalytically productive conformation. Conformational
changes after cleavage involves four disordered regions of the activation
domain, including the activation loop (residues 16-21), the autolysis loop
(residues 142-152), the oxyanion stabilizing loop (residues 184-194), and the
S1 entrance frame (residues 216-223) (Fig.
1A) (for reviews, see Refs.
1-3).Open in a separate windowFIGURE 1.Three-dimensional structure of uPA. A, overview of the
three-dimensional structure of the serine protease domain of active uPA,
displayed as ribbons. Depicted as sticks are the residues
Ile16, Asp194, and Ser195. The activation
domain, i.e. the activation loop (residues 16-21), the autolysis loop
(residues 142-152), the oxyanion stabilizing loop (residues 184-193), and the
S1 entrance frame (residues 216-223) are colored green. B, the
epitope of mAb-112, displayed on a surface presentation of the serine protease
domain of active uPA. Alanine substitution of residues depicted in
red resulted in a significant change in the affinity to mAb-112,
whereas alanine substitution of residues depicted in blue did not.
C, a close up view of the autolysis loop (residues Gly141
to Lys156) and residues implicated in the binding of mAb-112. All
figures were constructed with Pymol on the basis of the coordinates given in
the PDB entry 1C5W.Several proteases contribute to a variety of pathophysiological states,
thus stimulating considerable interest in the design of specific inhibitors
for pharmacological intervention. Nonetheless, classical development of small
molecule inhibitors of serine proteases has proved a daunting task, with only
limited success in engineering inhibitors with high affinity and specificity
for related proteases possessing conserved active site architecture and
P13 specificity
(4,
5). Thus far targeting zymogen
activation instead of the mature protease has been a greatly under-exploited
strategy in therapeutic regulation of protease activity. This approach
provides an opportunity to target more unique interaction surfaces, thereby
increasing inhibitor specificity, and ultimately offering novel inhibitory
mechanisms. In addition, a more efficient inhibition is expected by targeting
enzymes functioning high up in a catalytic cascade.A serine protease of particular relevance for pursuing therapeutic
intervention is urokinase-type plasminogen activator
(uPA),4 which
catalyzes the conversion of plasminogen to the active protease plasmin, which
in turn acts directly on the degradation of extracellular matrix proteins
(6). Abnormal expression of uPA
is implicated in tissue remodeling in several pathological conditions,
including rheumatoid arthritis, allergic vasculitis, and xeroderma
pigmentosum. In particular, uPA is central to the invasive capacity of
malignant tumors (6). As with
all trypsin-like proteases, uPA has a catalytic serine protease domain with
surface-exposed loops around residues 37, 60, 97, 110, 170, and 185. Besides
the catalytic domain, uPA has an amino-terminal extension consisting of a
kringle domain and an epidermal growth factor domain. The latter domain
functions in binding to the cell surface-anchored uPA receptor (uPAR)
(6). Several proteases
including plasmin (6),
glandular kallikrein (7),
matriptase (8), and hepsin
(9) can catalyze the activation
of the zymogen, pro-uPA.A number of inhibitors targeting the proteolytic activity of uPA have been
developed, such as small organochemical molecules, peptides, and monoclonal
antibodies, with a perspective on their use for elucidating the
pathophysiological functions of its various molecular interactions and
generating leads during drug development. The most specific inhibitors to date
appear to be those derived from antibodies and peptidyl inhibitors, which
utilize binding sites involving surface loops of uPA and extended exosite
interactions to drive selectivity and specificity (for reviews, see Refs.
4 and
5).Here we present evidence that targeting zymogen activation is an effective
means to regulate protease activity. This conclusion was realized through the
development and biochemical analysis of an inhibitory monoclonal antibody,
referred to as monoclonal antibody (mAb)-112, which not only delays cleavage
of pro-uPA but acts to stabilize the activated two-chain protease in a
non-catalytic conformation by restricting the conformational mobility of the
activation domain. Characterization of mAb-112 further provides new insights
into the flexibility of protease domains and uPA zymogen activation
mechanisms. Moreover, mAb-112 was shown to efficiently inhibit human tumor
cell intravasation, a step in the metastatic cascade in which activation of
pro-uPA was previously implicated as a key event
(10). 相似文献