Catalytic activity of an isolated domain of Na,K-ATPase expressed in Escherichia coli.
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Fusion proteins of glutathione-S-transferase and fragments from the large cytoplasmic domain of the sheep Na,K-ATPase alpha1-subunit were expressed in Escherichia coli. The Na,K-ATPase sequences begin at Ala345 and terminate at either Arg600 (DP600f), Thr610 (DP610f), Gly731 (DP731f), or Glu779 (DP779f). After affinity purification on glutathione-Sepharose, the fusion proteins were labeled with [alpha-32P]-2-N3-ATP, and incorporation of the radiolabel into the fusion proteins was measured by scintillation counting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kd values of 220-290 microM for 2-N3-ATP binding to the fusion proteins were obtained from the photolabeling experiments. Approximately 1 mol of 2-N3-ATP was calculated to be incorporated per mole of fusion protein after correction for photochemical incorporation efficiency. Labeling of all of the fusion proteins by 25 microM 2-N3-ATP was reduced in the presence of MgATP, Na2ATP, MgCl2, 2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and p-nitrophenylphosphate, and Ki values of 2-11 mM for Na2ATP, 0.2-5 mM for MgCl2, 0.1-5 mM for MgATP, and 20-300 microM for p-nitrophenylphosphate were calculated for these ligands. All of the fusion proteins catalyze the hydrolysis of p-nitrophenylphosphate. The reaction requires MgCl2 and is inhibited by inorganic phosphate, which is similar to the hydrolysis of p-nitrophenylphosphate by native Na,K-ATPase. Based on these observations, it appears that the soluble fragments from the large cytoplasmic domain of Na,K-ATPase expressed in bacterial cells are folded in an E2-like conformation and are likely to retain much of the native structure. (+info)
Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.
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The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol. (+info)
Activated memory CD4(+) T helper cells repopulate the intestine early following antiretroviral therapy of simian immunodeficiency virus-infected rhesus macaques but exhibit a decreased potential to produce interleukin-2.
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Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we performed a longitudinal study to determine the effect of antiretroviral therapy on the phenotype and functional potential of CD4(+) T cells repopulating intestinal mucosa in human immunodeficiency virus infection. Severe depletion of CD4(+) and CD4(+) CD8(+) T cells occurred in the intestinal mucosa during primary SIV infection. The majority of these cells were of activated memory phenotype. Phosphonate 9-[2-(phosphomethoxypropyl]adenine (PMPA) treatment led to a moderate suppression of intestinal viral loads and repopulation of intestinal mucosa by predominantly activated memory CD4(+) T-helper cells. This repopulation was independent of the level of viral suppression. Compared to preinfection values, the frequency of naive CD4(+) T cells increased following PMPA therapy, suggesting that new CD4(+) T cells were repopulating the intestinal mucosa. Repopulation by CD4(+) CD8(+) T cells was not observed in either jejunum or colon lamina propria. The majority of CD4(+) T cells repopulating the intestinal mucosa following PMPA therapy were CD29(hi) and CD11ahi. A subset of repopulating intestinal CD4(+) T cells expressed Ki-67 antigen, indicating that local proliferation may play a role in the repopulation process. Although the majority of repopulating CD4(+) T cells in the intestinal mucosa were functionally capable of providing B- and T-cell help, as evidenced by their expression of CD28, these CD4(+) T cells were found to have a reduced capacity to produce interleukin-2 (IL-2) compared to the potential of CD4(+) T cells prior to SIV infection. Persistent viral infection may play a role in suppressing the potential of repopulating CD4(+) T cells to produce IL-2. Hence, successful antiretroviral therapy should aim at complete suppression of viral loads in mucosal lymphoid tissues, such as intestinal mucosa. (+info)
Activity-dependent depression of GABAergic IPSCs in cultured hippocampal neurons.
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Short-term depression of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) evoked between pairs of cultured rat hippocampal neurons was investigated using dual whole cell patch-clamp recordings. Paired stimuli applied to the GABAergic neuron resulted in paired-pulse depression (PPD) of the second IPSC (IPSC2) at interpulse intervals from 25 to 2,000 ms. CGP 55845A, but not CGP 35348, reduced PPD marginally. Brief paired-pulse applications of exogenous GABA indicated that postsynaptic factors made only minimal contribution to PPD of IPSCs. IPSC1 and PPD was reduced on lowering [Ca2+]o and enhanced on increasing [Ca2+]o. The potassium-channel blocker 4-aminopyridine (4-AP), which increases presynaptic Ca2+ influx, enhanced IPSC1 and PPD. Chelation of residual Ca2+ in the GABAergic boutons with EGTA-AM enhanced PPD. Stimulation of the presynaptic neuron at frequencies (f) ranging from 2.5 to 80 Hz resulted in tetanic depression of IPSCs, which declined rapidly and reached a plateau depending on f and [Ca2+]o. CGP 55845A decreased tetanic depression in the first part of the train, but this could be overcome with continued stimulation. We show that GABAergic IPSCs are robustly depressed by paired-pulse stimulation in cultured hippocampal neurons. The depression of IPSCs is mainly independent of presynaptic GABAB receptors and could be caused by depletion of releasable vesicles. Depleted synapses recover with a slow time course, depending on factors that regulate [Ca2+]i in the GABAergic boutons. (+info)
Thermodynamics of the arginine kinase reaction.
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The effect of temperature, pH, free [Mg(2+)], and ionic strength on the apparent equilibrium constant of arginine kinase (EC 2.7.3.3) was determined. At equilibrium, the apparent K' was defined as [see text] where each reactant represents the sum of all the ionic and metal complex species. The K' at pH 7.0, 1.0 mM free [Mg(2+)], and 0. 25 M ionic strength was 29.91 +/- 0.59, 33.44 +/- 0.46, 35.44 +/- 0. 71, 39.64 +/- 0.74, and 45.19 +/- 0.65 (n = 8) at 40, 33, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy (DeltaH degrees') is -8.19 kJ mol(-1), and the corresponding standard apparent entropy of the reaction (DeltaS degrees') is + 2. 2 J K(-1)mol(-1) in the direction of ATP formation at pH 7.0, free [Mg(2+)] =1.0 mM, ionic strength (I) =0.25 M at 25 degrees C. We further show that the magnitude of transformed Gibbs energy (DeltaG degrees ') of -8.89 kJ mol(-1) is mostly comprised of the enthalpy of the reaction, with 7.4% coming from the entropy TDeltaS degrees' term (+0.66 kJ mol(-1)). Our results are discussed in relation to the thermodynamic properties of its evolutionary successor, creatine kinase. (+info)
Hydroxyurea enhances the activities of didanosine, 9-[2-(phosphonylmethoxy)ethyl]adenine, and 9-[2-(phosphonylmethoxy)propyl]adenine against drug-susceptible and drug-resistant human immunodeficiency virus isolates.
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We assessed the effects of hydroxyurea (HU) at a concentration of 50 microM on the in vitro activities of 2',3'-dideoxyinosine (ddI), 9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA), and 9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) against a wild-type human immunodeficiency virus (HIV) type 1 (HIV-1) laboratory isolate and a panel of five well-characterized drug-resistant HIV isolates. Fifty micromolar HU significantly increased the activities of ddI, PMEA, and PMPA against both the wild-type and the drug-resistant HIV-1 isolates. In fixed combinations, both ddI and PMEA were synergistic with HU against wild-type and drug-resistant viruses. (+info)
Phosphoantigen-reactive Vgamma9Vdelta2 T lymphocytes suppress in vitro human immunodeficiency virus type 1 replication by cell-released antiviral factors including CC chemokines.
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Vgamma9Vdelta2 T lymphocytes are broadly reactive against various intracellular pathogens and display both lytic and proliferative responses to human immunodeficiency virus (HIV)-infected cells. HIV infection of peripheral blood mononuclear cell cultures led to absolute increases in Vgamma9Vdelta2 T cells accompanied by decreased p24 levels. Strong gammadelta T cell activation with nonpeptidic mycobacterial phosphoantigens (TUBAg1 extract or synthetic isopentenyl pyrophosphate) resulted in potent inhibition of HIV replication through soluble released factors. Subsequent analyses showed that phosphoantigen-activated gammadelta T cells produced substantial amounts of beta-chemokines (macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, and regulated-on-activation, normal T-cell-expressed and -secreted beta-chemokine [RANTES]), which represent the natural ligand for the CCR5 HIV coreceptor. Accordingly, anti-beta-chemokine antibodies neutralized the inhibition of monocytotropic HIV strains by gammadelta T cell-released factors. Moreover, a T-tropic HIV strain using the CXCR4 coreceptor for virus entry was potently inhibited. Together, these data reveal that phosphoantigen-activated gammadelta T cells are an important source of CC chemokines and may suppress HIV replication through cell-released antiviral factors. (+info)
Increased stress right ventricular activity on dual isotope perfusion SPECT: a sign of multivessel and/or left main coronary artery disease.
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OBJECTIVES: This study sought to determine the anatomic and physiologic correlates of increased right ventricular (RV) activity on exercise single-photon emission computed tomography (SPECT) perfusion imaging in patients with coronary artery disease (CAD). BACKGROUND: Because SPECT perfusion imaging delineates relative myocardial blood flow, patients with global left ventricular (LV) hypoperfusion but normal RV perfusion may have increased relative RV tracer uptake as an indicator of multivessel CAD. METHODS: Rest thallium-201 and exercise 99mTc-sestamibi or 99mTc-tetrofosmin SPECT perfusion images were analyzed for peak RV and LV activity (RV:LV index) in 315 patients, including 240 patients with documented CAD, 39 patients with no significant CAD on arteriography, and a "normalcy" group of 36 patients with a low pre- and posttest probability of CAD. RESULTS: Resting RV:LV perfusion index ranged from 0.32 to 0.34 in each group, increasing to 0.36 with exercise in control and normalcy groups. CAD patients with the highest exercise RV:LV were those with severe left main CAD (or "left main equivalent"), with a lesser degree of proximal right CAD (0.51, n = 14, p < 0.001 vs. other groups). An exercise RV:LV >0.42 with a exercise:rest ratio >1.2 was present in 93% patients with this pattern of CAD, but was absent in 97% of the normalcy group, 92% of patients without significant angiographic CAD, and 100% of patients with proximal right CAD tighter than stenoses in the left system. CONCLUSIONS: Increased RV:LV activity exercise may occur in patients with acute RV strain, but is otherwise an indicator of exercise-induced RV:LV perfusion imbalance associated with severe CAD, particularly high-grade left main with less severe proximal right CAD. (+info)