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961.
Biermann J Lang D Gorboulev V Koepsell H Sindic A Schröter R Zvirbliene A Pavenstädt H Schlatter E Ciarimboli G 《American journal of physiology. Cell physiology》2006,290(6):C1521-C1531
Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (55 ± 5%, n = 10 and 63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (54 ± 5%, n = 14, and 31 ± 6%, n = 26) and PKA (16 ± 5%, n = 16, and 18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (16099 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride 相似文献
962.
Single plasma membrane K+ channel detection by using dual-color quantum dot labeling 总被引:1,自引:0,他引:1
Nechyporuk-Zloy V Stock C Schillers H Oberleithner H Schwab A 《American journal of physiology. Cell physiology》2006,291(2):C266-C269
K+ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K+ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K+ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K+ channel structure contrasts with the difficulties to visualize single K+ channel proteins in their physiological environment. We describe a method to identify single Ca2+-activated K+ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K+ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level. Ca2+-activated K+ channel; migration 相似文献
963.
The extracellular matrix (ECM) is composed of several families of macromolecular components: fibrous proteins such as collagens, type I collagen (COL1), type III collagen (COL3), fibronectin, elastin, and glycoconjugates such as proteoglycans and matrix glycoproteins. Their receptors on the cell membrane, most of which in the case of the ECM belong to the integrins, which are heterodimeric proteins composed of α and β chains. COL1 is the major fibrous collagen of bone, tendon, and skin; while COL3 is the more pliable collagen of organs like liver. Focus will not only be given to the regulation of synthesis of several fibrogenic parameters but also modulation of their degradation during growth factor‐induced tissue fibrosis and cancer development. Evidence will be provided that certain tissues, which undergo fibrosis, also become cancerous. Why does there exist a divergency between tissues, which undergo frank fibrosis as an endpoint, and those tissues that undergo fibrosis and subsequently are susceptible to carcinogenicity; resulting from the etiological factor(s) causing the initial injury? For example, why does a polyvinyl alcohol (PVA) sponge implant become encapsulated and filled with fibrous tissue then fibrosis tissue growth stops? Why does the subcutaneous injection of a fibrogenic growth factor cause a benign growth and incisional wounding results in fibrosis and ultimately scarring? There are many examples of tissues, which undergo fibrosis as a prerequisite to carcinogenesis. Is there a cause‐effect relationship? If you block tissue fibrosis in these precancerous tissues, would you block cancer formation? What are the molecular targets for blocking fibrosis and ultimately carcinogenesis? How can oligo decoys may be used to attenuate carcinogenesis and which oligo decoys specifically attenuate fibrogenesis as a prelude to carcinogenesis? What are other molecular targets for oligo decoy therapy in carcinogenesis? J. Cell. Biochem. 97: 1161–1174, 2006. © 2006 Wiley‐Liss, Inc. 相似文献
964.
Zengin E Chalajour F Gehling UM Ito WD Treede H Lauke H Weil J Reichenspurner H Kilic N Ergün S 《Development (Cambridge, England)》2006,133(8):1543-1551
Here, we report the existence of endothelial precursor (EPC) and stem cells in a distinct zone of the vascular wall that are capable to differentiate into mature endothelial cells, hematopoietic and local immune cells, such as macrophages. This zone has been identified to be localized between smooth muscle and adventitial layer of human adult vascular wall. It predominantly contains CD34-positive (+) but CD31-negative (-) cells, which also express VEGFR2 and TIE2. Only few cells in this zone of the vascular wall are positive for CD45. In a ring assay using the fragments of human internal thoracic artery (HITA), we show here that the CD34+ cells of the HITA-wall form capillary sprouts ex vivo and are apparently recruited for capillary formation by tumor cells. New vessels formed by these vascular wall resident EPCs express markers for angiogenically activated endothelial cells, such as CEACAM1, and also for mature endothelial cells, such as VE-cadherin or occludin. Vascular wall areas containing EPCs are found in large and middle sized arteries and veins of all organs studied here. These data suggest the existence of a ;vasculogenic zone' in the wall of adult human blood vessels, which may serve as a source for progenitor cells for postnatal vasculogenesis, contributing to tumor vascularization and local immune response. 相似文献
965.
Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, we identified piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. We further show that loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a muscle-derived attractant. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process. 相似文献
966.
Alderwick LJ Dover LG Seidel M Gande R Sahm H Eggeling L Besra GS 《Glycobiology》2006,16(11):1073-1081
The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis. 相似文献
967.
Gürleyen H Christiansen H Tello K Dudas J Hermann RM Rave-Fränk M Hess CF Ramadori G Saile B 《Radiation and environmental biophysics》2009,48(1):85-94
This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes
to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides
prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased
in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of
caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes
to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may
play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration
depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB.
H. Gürleyen and H. Christiansen contributed equally to this work. 相似文献
968.
Petra Hänzelmann Jan U. Dahl Jochen Kuper Alexander Urban Ursula Müller‐Theissen Silke Leimkühler Hermann Schindelin 《Protein science : a publication of the Protein Society》2009,18(12):2480-2491
Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent. 相似文献
969.
Carla C. C. R. de Carvalho Lukas Y. Wick Hermann J. Heipieper 《Applied microbiology and biotechnology》2009,82(2):311-320
Rhodococcus erythropolis was found to utilize C5 to C16 n-alkane hydrocarbons as sole source of carbon and energy when growing as planktonic or biofilm cells attached to polystyrene
surfaces. Growth rates on even numbered were two- to threefold increased relatively to odd-numbered n-alkanes and depended on the chain length of the n-alkanes. C10-, C12-, C14-, and C16-n-alkanes gave rise to two- to fourfold increased maximal growth rates relative to C5- to C9-hydrocarbons. In reaction to the
extremely poor water solubility of the n-alkanes, both planktonic and biofilm cells exhibited distinct adaptive changes. These included the production of surface
active compounds and substrate-specific modifications of the physicochemical cell surface properties as expressed by the microbial
adhesion to hydrocarbon- and contact angle-based hydrophobicity, as well as the zeta potential of the cells. By contrast,
n-alkane-specific alterations of the cellular membrane composition were less distinct. The specificity of the observed autecological
changes suggest that R. erythropolis cells may be used in the development and application of sound biocatalytic processes using n-alkanes as substrates or substrate reservoirs or for target-specific bioremediation of oils and combustibles, respectively. 相似文献
970.
Gilles A. Spoden Dieter Morandell Daniela Ehehalt Marc Fiedler Pidder Jansen‐Dürr Martin Hermann Werner Zwerschke 《Journal of cellular biochemistry》2009,107(2):293-302
Pyruvate kinase M2 (M2‐PK) controls the rate‐limiting step at the end of the glycolytic pathway in normal proliferating and tumor cells. Other functions of M2‐PK in addition to its role in glycolysis are little understood. The aim of this study was to identify new cellular interaction partners of M2‐PK in order to discover novel links between M2‐PK and cellular functions. Here we show that the SUMO‐E3 ligase protein PIAS3 (inhibitor of activated STAT3) physically interacts with M2‐PK and its isoenzyme M1‐PK. Moreover, we demonstrate that endogenous SUMO‐1‐M2‐PK conjugates exist in mammalian cells. Furthermore, we show that transient expression of PIAS3 but not the RING domain mutant PIAS3 (C299S, H301A) is consistent with nuclear localization of M2‐PK and PIAS3 and M2‐PK partially co‐localize in the nucleus of these cells. This study suggests a link between PIAS3 and nuclear pyruvate kinase. J. Cell. Biochem. 107: 293–302, 2009. © 2009 Wiley‐Liss, Inc. 相似文献