Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin.
Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory. (+info)
Relationship between L-type Ca2+ current and unitary sarcoplasmic reticulum Ca2+ release events in rat ventricular myocytes.
1. The time courses of Ca2+ current and Ca2+ spark occurrence were determined in single rat ventricular myocytes voltage clamped with patch pipettes containing 0.1 microM fluo-3. Acquisition of line-scan images on a laser scanning confocal microscope was synchronized with measurement of Cd2+-sensitive Ca2+ currents. In most cells, individual Ca2+ sparks were observed by reducing Ca2+ current density with nifedipine (0.1-8 microM). 2. Ca2+ sparks elicited by depolarizing voltage-clamp pulses had a peak [Ca2+] amplitude of 289 +/- 3 nM with a decay half-time of 20.8 +/- 0.2 ms and a full width at half-maximum of 1.40 +/- 0.03 microm (mean +/- s. e.m., n = 345), independent of the membrane potential. 3. The time between the beginning of a depolarization and the initiation of each Ca2+ spark was calculated and data were pooled to construct waiting time histograms. Exponential functions were fitted to these histograms and to the decaying phase of the Ca2+ current. This analysis showed that the time constants describing Ca2+ current and Ca2+ spark occurrence at membrane potentials between -30 mV and +30 mV were not significantly different. At +50 mV, in the absence of nifedipine, the time constant describing Ca2+ spark occurrence was significantly larger than the time constant of the Ca2+ current. 4. A simple model is developed using Poisson statistics to relate macroscopic Ca2+ current to the opening of single L-type Ca2+ channels at the dyad junction and to the time course of Ca2+ spark occurrence. The model suggests that the time courses of macroscopic Ca2+ current and Ca2+ spark occurrence should be closely related when opening of a single L-type Ca2+ channel initiates a Ca2+ spark. By comparison with the data, the model suggests that Ca2+ sparks are initiated by the opening of a single L-type Ca2+ channel at all membrane potentials encountered during an action potential. (+info)
Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+].
The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways. (+info)
Diverse oxygenations catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp. Strain CA10.
Carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas sp. strain CA10 is a multicomponent enzyme that catalyzes the angular dioxygenation of carbazole, dibenzofuran, and dibenzo-p-dioxin. It was revealed by gas chromatography-mass spectrometry and 1H and 13C nuclear magnetic resonance analyses that xanthene and phenoxathiin were converted to 2,2',3-trihydroxydiphenylmethane and 2,2',3-trihydroxydiphenyl sulfide, respectively. Thus, for xanthene and phenoxathiin, angular dioxygenation by CARDO occurred at the angular position adjacent to the oxygen atom to yield hetero ring-cleaved compounds. In addition to the angular dioxygenation, CARDO catalyzed the cis dihydroxylation of polycyclic aromatic hydrocarbons and biphenyl. Naphthalene and biphenyl were converted by CARDO to cis-1, 2-dihydroxy-1,2-dihydronaphthalene and cis-2,3-dihydroxy-2, 3-dihydrobiphenyl, respectively. On the other hand, CARDO also catalyzed the monooxygenation of sulfur heteroatoms in dibenzothiophene and of the benzylic methylenic group in fluorene to yield dibenzothiophene-5-oxide and 9-hydroxyfluorene, respectively. These results indicate that CARDO has a broad substrate range and can catalyze diverse oxygenation: angular dioxygenation, cis dihydroxylation, and monooxygenation. The diverse oxygenation catalyzed by CARDO for several aromatic compounds might reflect the differences in the binding of the substrates to the reaction center of CARDO. (+info)
Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells.
The mechanism of Ni2+ block of the Na+/Ca2+ exchanger was examined in Sf 9 cells expressing the human heart Na+/Ca2+ exchanger (NCX1-NACA1). As predicted from the reported actions of Ni2+, its application reduced extracellular Na+-dependent changes in intracellular Ca2+ concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied by measured 63Ni2+ entry. The 63Ni2+ entry was observed in Sf 9 cells expressing the Na+/Ca2+ exchanger but not in control cells. The established sequential transport mechanism of the Na+/Ca2+ exchanger could be compatible with these results if one of the two ion translocation steps is blocked by Ni2+ and the other permits Ni2+ translocation. We conclude that, because Ni2+ entry was inhibited by extracellular Ca2+ and enhanced by extracellular Na+, the Ca2+ translocation step moved Ni2+, whereas the Na+ translocation step was inhibited by Ni2+. A model is presented to discuss these findings. (+info)
Structural insight into a quinolone-topoisomerase II-DNA complex. Further evidence for a 2:2 quinobenzoxazine-mg2+ self-assembly model formed in the presence of topoisomerase ii.
Quinobenzoxazine A-62176, developed from the antibacterial fluoroquinolones, is active in vitro and in vivo against murine and human tumors. It has been previously claimed that A-62176 is a catalytic inhibitor of mammalian topoisomerase II that does not stabilize the cleaved complex. However, at low drug concentrations and pH 6-7, we have found that A-62176 can enhance the formation of the cleaved complex at certain sites. Using a photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+ self-assembly model was previously proposed, in which one drug molecule intercalates into the DNA helix and the second drug molecule is externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments between psorospermin and A-62176, as well as between psorospermin and A-62176 and norfloxacin, are consistent with this model and provide the first evidence that this 2:2 quinobenzoxazine-Mg2+ complex is assembled in the presence of topoisomerase II. These results also have parallel implications for the mode of binding of the quinolone antibiotics to the bacterial gyrase-DNA complex. (+info)
Thalidomide increases both intra-tumoural tumour necrosis factor-alpha production and anti-tumour activity in response to 5,6-dimethylxanthenone-4-acetic acid.
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), synthesized in this laboratory and currently in phase I clinical trial, is a low molecular weight inducer of tumour necrosis factor-alpha (TNF-alpha). Administration of DMXAA to mice with established transplantable tumours elicits rapid vascular collapse selectively in the tumour, followed by extensive haemorrhagic necrosis mediated primarily through the production of TNF-alpha. In this report we have investigated the synthesis of TNF-alpha mRNA in hepatic, splenic and tumour tissue. Co-administration of thalidomide with DMXAA increased anti-tumour activity and increased intra-tumoural TNF-alpha production approximately tenfold over that obtained with DMXAA alone. Thalidomide increased splenic TNF-alpha production slightly but significantly decreased serum and hepatic levels of TNF-alpha induced with DMXAA. Lipopolysaccharide (LPS) induced 300-fold higher serum TNF-alpha than did DMXAA at the maximum tolerated dose, but induced similar amounts of TNF-alpha in spleen, liver and tumour. Splenic TNF-alpha activity induced with LPS was slightly increased with thalidomide, but serum and liver TNF-alpha levels were suppressed. Thalidomide did not increase intra-tumoural TNF-alpha production induced with LPS, in sharp contrast to that obtained with DMXAA. While thalidomide improved the anti-tumour response to DMXAA, it had no effect on the anti-tumour action of LPS that did not induce a significant growth delay or cures against the Colon 38 tumour. The increase in the anti-tumour action by thalidomide in combination with DMXAA corresponded to an increase in intra-tumoural TNF-alpha production. Co-administration of thalidomide may represent a novel approach to improving selective intra-tumoural TNF-alpha production and anti-tumour efficacy of DMXAA. (+info)
Morphological changes and detachment of adherent cells induced by p122, a GTPase-activating protein for Rho.
We recently cloned a novel signaling molecule, p122, that shows a GTPase-activating activity specific for Rho and the ability to enhance the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of phospholipase C delta1 in vitro. Here we analyzed the in vivo function of p122. Microinjection of the GTPase-activating domain of p122 suppressed the formation of stress fibers and focal adhesions induced by lysophosphatidic acid, suggesting a GTPase-activating activity for Rho as in in vitro. Transfection of p122 also induced the disassembly of stress fibers and the morphological rounding of various adherent cells. Analyses using deletion and point mutants demonstrated that the GTPase-activating domain of p122 is responsible for the morphological changes and detachment and that arginine residues at positions 668 and 710 and a lysine residue at position 706 in the GTPase-activating domain are essential. Using Fluo-3-based Ca2+ microscopy, we found that p122 evoked a rapid elevation of intracellular Ca2+ levels, suggesting that p122 stimulates the phosphatidylinositol 4, 5-bisphosphate-hydrolyzing activity of phospholipase C delta1. These results demonstrate that p122 synergistically functions as a GTPase-activating protein specific for Rho and an activator of phospholipase C delta1 in vivo and induces morphological changes and detachment through cytoskeletal reorganization. (+info)