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Andersen GØ Skomedal T Enger M Fidjeland A Brattelid T Levy FO Osnes JB 《American journal of physiology. Heart and circulatory physiology》2004,286(4):H1354-H1360
We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway. 相似文献
74.
Ahmad T Ugarph-Morawski A Li J Bileviciute-Ljungar I Finn A Ostenson CG Kreicbergs A 《Regulatory peptides》2004,119(1-2):61-67
We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia. 相似文献
75.
Yu XY Finn J Hill JM Wang ZG Keith D Silverman J Oliver N 《Bioorganic & medicinal chemistry letters》2004,14(5):1339-1342
We have identified a series of spirocyclic furan and pyrrolidine inhibitors of Enterococcus faecalis and Staphylococcus aureus phenylalanyl-tRNA synthetases. The most potent analogue 1b showed IC50=5 nM (E. faecalis PheRS) and IC50=2 nM (S. aureus PheRS) with high selectivity over the human enzyme. The crystal X-ray structure of analogue 1b was determined. 相似文献
76.
Gårdsvoll H Werner F Søndergaard L Danø K Ploug M 《Protein expression and purification》2004,34(2):284-295
The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography. 相似文献
77.
NMR diffusion as a novel tool for measuring the association constant between cyclodextrin and guest molecules 总被引:4,自引:0,他引:4
In this paper we introduce the use of diffusion measurements by nuclear magnetic resonance (NMR) spectroscopy for determining association constants of weak and very weak interactions between cyclodextrin and guest molecules, as long as both the free and complexed guest molecules are soluble to an extent that allows good sensitivity in the NMR experiment. The experimental setup and data analysis is discussed for three different guest molecules: L-phenylalanine, L-leucine and L-valine, representing different strengths of interaction. The underlying assumptions are discussed and the scope of the method (range of K(a) values, requirements to the guest molecule) are discussed. The method's main advantage is its general applicability independent of chromogenic or electrochemical properties of the guest molecule. Whereas calorimetric methods that exhibit a similar generality, are applicable mainly to strong interactions, NMR diffusion measurements are applicable to weaker interactions down to the theoretical limit of 1 M(-1), the upper limit for K(a) values to be determined by it is approximately 200. A further advantage of the method is the low amount of sample needed. The method is in principle applicable to any case of molecular recognition between a host and guest molecule leading to weak interactions. 相似文献
78.
Vitamin K uptake in hepatocytes and hepatoma cells 总被引:1,自引:0,他引:1
Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation. 相似文献
79.
80.
Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck 总被引:4,自引:0,他引:4
Schweimer K Hoffmann S Bauer F Friedrich U Kardinal C Feller SM Biesinger B Sticht H 《Biochemistry》2002,41(16):5120-5130
Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck. 相似文献