Magnesium ion-induced changes in the binding mode of adenylates to chloroplast coupling factor 1.
(17/29202)
The effect of Mg2+ on the binding of adenylates to isolated chloroplast coupling factor 1 (CF1) was studied using CD spectrometry and ultrafiltration. At adenylate concentrations smaller than 100 muM, one mole of CF1 binds three moles of ATP (or ADP) regardless of the presence of Mg2+. In the presence of Mg2+, the first two ATP's bind to CF1 independently with the same binding constant of 2.5 X 10(-1) muM-1, then the third ATP binds with a much higher affinity of 10 muM-1. In the absence of Mg2+, the first ATP binds to CF1 with a binding constant of 2.5 X 10(-1) muM-1 then the other two ATP's bind less easily with the same binding constant of 4.0 X 10(-2) muM-1. The binding mode of ADP to CF1 is quite similar to that of ATP. In the presence of Mg2+, the binding constants of the first two ADP's are both 7.6 X 10(-2) muM-1, that of the third ADP being 4.0 muM-1. In the absence of Mg2+, the binding constant of the first ADP is 7.6 X 10(-2) muM-1, the constants of the other two ADP's both being 4.0 X 10(-2) muM-1. AMP caused a negligible change in CD. (+info)
Myocardial oxygenation during high work states in hearts with postinfarction remodeling.
(18/29202)
BACKGROUND: Postinfarction left ventricular remodeling (LVR) is associated with reductions in myocardial high-energy phosphate (HEP) levels, which are more severe in animals that develop overt congestive heart failure (CHF). During high work states, further HEP loss occurs, which suggests demand-induced ischemia. This study tested the hypothesis that inadequate myocyte oxygen availability is the basis for these HEP abnormalities. METHODS AND RESULTS: Myocardial infarction was produced by left circumflex coronary artery ligation in swine. Studies were performed in 20 normal animals, 14 animals with compensated LVR, and 9 animals with CHF. Phosphocreatine (PCr)/ATP was determined with 31P NMR and deoxymyoglobin (Mb-delta) with 1H NMR in myocardium remote from the infarct. Basal PCr/ATP tended to be decreased in postinfarct hearts, and this was significant in animals with CHF. Infusion of dobutamine (20 microg x kg-1 x min-1 IV) caused doubling of the rate-pressure product in both normal and LVR hearts and resulted in comparable significant decreases of PCr/ATP in both groups. This decrease in PCr/ATP was not associated with detectable Mb-delta. In CHF hearts, rate-pressure product increased only 40% in response to dobutamine; this attenuated response also was not associated with detectable Mb-delta. CONCLUSIONS: Thus, the decrease of PCr/ATP during dobutamine infusion is not the result of insufficient myocardial oxygen availability. Furthermore, in CHF hearts, the low basal PCr/ATP and the attenuated response to dobutamine occurred in the absence of myocardial hypoxia, indicating that the HEP and contractile abnormalities were not the result of insufficient oxygen availability. (+info)
[3H]gemcitabine uptake by nucleoside transporters in a human head and neck squamous carcinoma cell line.
(19/29202)
Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma membrane. In the present study, we examined the role of these transporters in the accumulation of gemcitabine by a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitibine was compared with that of [3H]uridine and [3H]formycin B in the parent cell line (HN-5a) and in a gemcitabine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equilibrative, inhibitor-sensitive) transporter subtype; less than 10% of the influx of [3H]formycin B or [3H]uridine was mediated by the ei (equilibrative inhibitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]Gemcitabine (10 microM) entered these cells via both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]formycin B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitabine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitabine. These results show that gemcitabine is a substrate for both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitabine resistance of the GEM-8e cells cannot be attributed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted because this system has the potential to significantly affect the clinical efficacy of gemcitabine. (+info)
Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.
(20/29202)
We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability. (+info)
Mechanical stimulation of starfish sperm flagella.
(21/29202)
1. The responses of starfish sperm flagella to mechanical stimulation with a microneedle were analysed. Flagellar movement was recorded by high-speed microcinematography and by stroboscopic observation. 2. The amplitude of the bending wave of a flagellum was restricted over its entire length when the microneedle was brought near to the flagellum at its proximal region. Beyond the restricted part, the amplitude of the wave, and the bend angle, became smaller than those of a normally beating flagellum, while the curvature was practically unchanged. 3. When the tip of the microneedle was in contact with the flagellum, propagation of the bending wave beyond the microneedle was inhibited. The part of the flagellum between the base and the microneedle continued beating in some cases and stopped beating in other cases. The flagellum beyond the arrested part stopped beating and remained straight. When the microneedle was removed, the bending wave which existed in the part of the flagellum proximal to the microneedle, or the wave which was passively formed de novo at the time of the removal of the microneedle, propagated over the arrested part towards the tip. 4. A flagellum amputated by a microneedle in a medium containing ATP continued beating with a small amplitude, small curvature, small bend angle and low frequency. When the amputated flagellum was passively bent by a microneedle at the region near the point of amputation, this bend propagated towards the tip with a constant bend angle. 5. The beating frequency of the flagellum could be modulated by the application of a rhythmic external force generated by vibrating a microneedle near the flagellum. The beating was completely synchronized with vibration of the microneedle in the frequency range from 23 Hz to 43 Hz. (+info)
2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages II. Dissociation of the inhibitory effects of 2-deoxyglucose on phagocytosis and ATP generation.
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Macrophages incubated in 2-deoxy-D-glucose (2-dG)-containing medium showed a marked decrease in cellular ATP content, and were unable to ingest IgG- and complement-coated erythrocytes via the corresponding membrane receptors for these ligands. However, the inhibitory effects of 2-dG on Fc- and C3 receptor-mediated phagocytosis were not a consequence of lowered macrophage ATP levels since addition of glucose or mannose to the culture medium restored the capacity of the macrophages to ingest IgG- and C3-coated particles without increasing ATP levels. These results indicate that Fc- and C3 receptor-mediated phagocytosis (opsonin dependent) differs qualitatively from the ingestion of latex and zymosan particles (opsonin independent); they suggest that the same regulatory molecules govern the responses of phagocytic cells to signals initiated by both the Fc and C3 receptors. The possibility that these molecules are regulated by glycosylation is discussed. (+info)
2,3 diphosphoglycerate in Parkinson's disease.
(23/29202)
The red cell 2,3 DPG, the most important factor for oxygen delivery in the tissues, was found to be increased in Parkinsonism patients compared with controls. The aging process seems not to be a factor in the increased 2,3 DPG concentration. Other factors relevant to raised 2,3 DPG level such as physical activity, increased oxygen requirements, and metabolic changes are discussed. (+info)
Bound forms of Ca taken up by the synaptic plasma membrane.
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Temperature dependent Ca-binding by the synaptic plasma membrane was increased in the presence of ATP and Mg++. Apparent Km for ATP was about 2.8 X 10(-5) M and optimal concentration of Mg++ was 2 mM in the presence of 2 mM ATP. After preincubation with nonradioactive Ca++, ATP and Mg++ to attain a steady state, addition of 45Ca resulted in remarkable labelling of the membrane, indicating rapid turnover of most of the membrane bound Ca. The presence of oxalate (60 mM) greatly increased Ca up-take on prolonged incubation. The Ca uptake in presence and absence of oxalate had similar substrate specificity and was similarly influenced by various monovalent cations. Furthermore, activities for Ca-uptake in the presence and absence of oxalate could not be separated by sucrose density gradient centrifugation of the synaptic plasma membrane fraction. Accordingly, it was considered that Ca++ in the medium was taken up by surface of the membrane, ATP- and temperature-dependently and then transferred into a cavity where the Ca-oxalate complex is formed. (+info)