Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate. (+info)
(2/1589) Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter.
Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins. (+info)
(3/1589) Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition.
Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca2+ dependence of force and dichroism. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01 of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM, n = 5) of the value at pCa 9. 2, and force was 0.51 +/- 0.04 (+/- SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+]. (+info)
(4/1589) Resolution of fluorescence correlation measurements.
The resolution limit of fluorescence correlation spectroscopy for two-component solutions is investigated theoretically and experimentally. The autocorrelation function for two different particles in solution were computed, statistical noise was added, and the resulting curve was fitted with a least squares fit. These simulations show that the ability to distinguish between two different molecular species in solution depends strongly on the number of photons detected from each particle, their difference in size, and the concentration of each component in solution. To distinguish two components, their diffusion times must differ by at least a factor of 1.6 for comparable quantum yields and a high fluorescence signal. Experiments were conducted with Rhodamine 6G and Rhodamine-labeled bovine serum albumin. The experimental results support the simulations. In addition, they show that even with a high fluorescence signal but significantly different quantum yields, the diffusion times must differ by a factor much bigger than 1.6 to distinguish the two components. Depending on the quantum yields and the difference in size, there exists a concentration threshold for the less abundant component below which it is not possible to determine with statistical means alone that two particles are in solution. (+info)
(5/1589) A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes.
1. The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton-coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy-SNARF-1 fluorescence in combination with whole-cell microspectrofluorimetry or confocal microscopy. 2. In Hepes-buffered Tyrode solution, application of the dipeptide Phe-Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole. 3. In the presence of CO2/HCO3--buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less. 4. Following application of the carbonic anhydrase inhibitor acetazolamide (100 microM) in the presence of CO2/HCO3- buffer, addition of Phe-Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole. 5. We conclude that acid influx due to proton-coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration. (+info)
(6/1589) In vivo significance of ICAM-1--dependent leukocyte adhesion in early corneal angiogenesis.
PURPOSE: Numerous investigations have stressed the significance of leukocytes in early angiogenesis. Leukocytes invade the cornea, and the location of their extravasation corresponds to the site of vessel ingrowth. The interactions between leukocytes and vascular endothelium are mediated by various proteins, including adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). In this study, the role of ICAM-1 during early corneal angiogenesis was evaluated in vivo. METHODS: Corneal neovascularization was induced in New Zealand White rabbits by use of intrastromal pellets containing 750 ng vascular endothelial growth factor (VEGF). The fluorescent dye rhodamine 6G was used to stain leukocytes in vivo. Leukocyte adhesion and vessel growth were quantified in vivo by high-resolution fluorescence angiography. To inhibit ICAM-1 interactions a microemulsion containing anti-ICAM-1 antibody was applied topically. RESULTS: Limbal vessels showed increased leukocyte adhesion 24 hours after pellet implantation: The number of rolling and sticking leukocytes was significantly increased compared with the number in control animals (P < 0.01). Treatment with anti-ICAM-1 antibody resulted in reduced leukocyte sticking and increased leukocyte rolling. The area covered by new blood vessels was significantly diminished in eyes treated with anti-ICAM-1 (P < 0.05). CONCLUSIONS: The results support the hypothesis that ICAM-1-mediated leukocyte adhesion is a key event in early angiogenesis. This model may serve for investigation of the significance of adhesion molecules by in vivo observation and quantification. (+info)
(7/1589) Inhibition of IL-6 and IL-8 induction from cultured rheumatoid synovial fibroblasts by treatment with aurothioglucose.
Gold compounds have long been used in the treatment of rheumatoid arthritis (RA). However, their actions in RA have not been clarified. In this study, we examined the effect of one of the monovalent gold compounds, aurothioglucose (AuTG), on the IL-1-induced production of IL-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) from rheumatoid synovial fibroblasts (RSF) isolated from three RA patients. IL-6 and IL-8 induction but not GM-CSF induction was inhibited in most of the RSF after pretreatment with AuTG. Since gene expression of these cytokines is known to be under the control of a common transcription factor, NF-kappaB, the effect of AuTG on the cellular localization of NF-kappaB (p65 subunit) and on NF-kappaB-DNA binding was examined. Although AuTG treatment did not prevent NF-kappaB nuclear translocation, AuTG blocked the DNA-binding activity of NF-kappaB when examined in vitro. Morphologically, both metal-specific cell staining using p-dimethylaminobenzylidene rhodamine and transmission electron microscopic examinations demonstrated the accumulation of metal gold in the cytoplama and some organella (mitochondria and lysosomes) of the AuTG-treated RSF. These results indicate that one of the anti-rheumatic actions of AuTG might be through its inhibitory action on NF-kappaB. (+info)
(8/1589) Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist.
Partial agonists are effective tools for advancing development of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native pancreatic acinar cells and Chinese hamster ovary cells expressing CCK receptor (CHO-CCKR). We developed and characterized a novel fluorescent partial agonist, rhodamine-Gly-[(Nle28, 31)CCK-26-32]-phenethyl ester, that binds specifically and with high affinity to CCK receptors. Such analogs are fully efficacious pancreatic acinar cell secretagogues without supramaximal inhibition that mobilize intracellular calcium with little or no increase in phospholipase C (PLC) activity. Despite minimal phosphorylation of CCK receptors in response to this partial agonist, receptor trafficking was the same as that observed with full agonist (CCK). This included normal internalization via clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the surface of pancreatic acinar cells. Also, as with CCK-occupied receptor, fluorescence recovery after photobleaching of partial agonist-occupied receptor on the acinar cell surface demonstrated a marked temperature-dependent slowing of its rate of diffusion. This was similarly associated with resistance to acid-induced dissociation of ligand. Thus some key molecular regulatory mechanisms for CCK receptor internalization and insulation may be initiated by cellular signaling cascades that are not dependent on PLC activation or receptor phosphorylation. (+info)