Dactinomycin Impairs Cellular Respiration and Reduces Accompanying ATP Formation

The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin

Effects of Cisplatin on Mitochondrial Function in Jurkat Cells

In this work, we measured the effects of pharmacological concentrations of cisplatin (cis-diaminedichloroplatinum II) on mitochondrial function, cell viability, and DNA fragmentation in Jurkat cells. The exposure of cells to 0-25 microM cisplatin for 3 h had no immediate effect on cellular mitochondrial oxygen consumption, measured using a palladium-porphyrin oxygen sensing phosphor. Similarly, the cell viability as measured by trypan blue staining was unchanged immediately following exposure to the drug, and no small DNA fragments, characteristic of drug-induced apoptosis, appeared. At 24 h after exposure to cisplatin, cellular respiration and viability decreased relative to controls and the amount of small DNA fragments, measured using quantitative agarose gel electrophoresis, was proportional to the concentration of cisplatin present during the drug exposure period. The small DNA fragments showed the banding pattern (with a spacing of approximately 300 bp) characteristic of drug-induced cell death by apoptosis. The changes in respiration and DNA fragmentation correlated linearly with the amount of platinum bound to DNA, determined by atomic absorption spectroscopy immediately following drug exposure. The oxygen consumption by beef heart mitochondria was not affected 0-24 h after exposure to 25 microM cisplatin or to solutions containing the monoaquated form of the drug, suggesting that the drug does not attack the mitochondrial respiratory chain directly. Cells exposed to the peptide benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone, which blocks apoptosis by the caspase pathway, showed a decrease in cisplatin-induced DNA fragmentation but not in the impairment of cellular respiration. Thus, although apoptosis is caspase-dependent, the impairment of cellular respiration is independent of the caspase system. Collectively, these results suggest that alteration in mitochondrial function is a secondary effect of cisplatin cytotoxicity in Jurkat cells

Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture

Objectives: This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures.Methods: Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Results: In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min-1 mg-1 (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min-1 per 107 cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. Conclusion: The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation

Cold Inactivation of l-Threonine Deaminase from Rhodospirillum rubrum

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66171/1/j.1432-1033.1971.tb01491.x.pd

A technique for the determination of protein concentration by neutron activation analysis of silver binding

A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific characteristics of each protein. Silver binding has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBH 4 , HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negligible amounts of silver remained bound to the protein. The stoichiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that the binding of silver is not specific to any charged or polar groups on these proteins and may, therefore, provide a means of determination of the concentration of protein that has general application for all proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43109/1/10967_2005_Article_BF02037279.pd

The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

Complete details of the thermodynamics and molecular mechanisms of ATP synthesis/hydrolysis and muscle contraction are offered from the standpoint of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. The manifold fundamental consequences and mechanistic implications of the unified theory for oxidative phosphorylation and muscle contraction are explained. The consistency of current mechanisms of ATP synthesis and muscle contraction with experiment is assessed, and the novel insights of the unified theory are shown to take us beyond the binding change mechanism, the chemiosmotic theory and the lever arm model. It is shown from first principles how previous theories of ATP synthesis and muscle contraction violate both the first and second laws of thermodynamics, necessitating their revision. It is concluded that the new paradigm, ten years after making its first appearance, is now perfectly poised to replace the older theories. Finally, applications of the unified theory in cell life and cell death are outlined and prospects for future research are explored. While it is impossible to cover each and every specific aspect of the above, an attempt has been made here to address all the pertinent details and what is presented should be sufficient to convince the reader of the novelty, originality, breakthrough nature and power of the unified theory, its manifold fundamental consequences and mechanistic implications, and its applications in health and disease

Multi-Parametric Analysis and Modeling of Relationships between Mitochondrial Morphology and Apoptosis

Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis