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.

The phosphorescence oxygen analyzer as a screening tool for disorders with impaired lymphocyte bioenergetics.

This study aimed to show the feasibility of using the phosphorescence oxygen analyzer to screen for clinical disorders with impaired cellular bioenergetics. [O(2)] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, O(2) consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics.

Measurement of oxygen consumption by murine tissues in vitro.

Introduction: A novel in vitro system was developed to measure O₂ consumption by murine tissues over several hours.

Methods: Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O₂:5% CO₂. The specimens were incubated at 37 °C in the buffer, continuously gassed with O₂:CO₂ (95:5).

Biocompatibility of calcined mesoporous silica particles with cellular bioenergetics in murine tissues.

A novel in vitro system was developed to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues. O(2) consumption by lung, liver, kidney, spleen, and pancreatic tissues was unaffected by exposure to 200 μg/mL MCM41-cal or SBA15-cal for several hours. Normal tissue histology was confirmed by light microscopy.

Cannabinoids inhibit cellular respiration of human oral cancer cells.

Background And Purpose: The primary cannabinoids, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and Delta(8)-tetrahydrocannabinol (Delta(8)-THC) are known to disturb the mitochondrial function and possess antitumor activities. These observations prompted us to investigate their effects on the mitochondrial O(2) consumption in human oral cancer cells (Tu183). This epithelial cell line overexpresses bcl-2 and is highly resistant to anticancer drugs.

Oxygen measurements via phosphorescence.

Accurate measurements of dissolved O(2) as a function of time have numerous chemical and biological applications. The Pd (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was used for this purpose. Detection is based on the principle that the phosphorescence of this oxygen probe is inversely related to dissolved O(2) (O(2) quenches the phosphorescence).

Cannabinoids inhibit the respiration of human sperm.

Objective: To investigate the effects of the psychotropic compounds Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and Delta(8)-tetrahydrocannabinol (Delta(8)-THC) on sperm mitochondrial O(2) consumption (respiration).

Setting: State University of New York Upstate Medical University, Syracuse, New York.

Patient(s): Forty-one men who visited the andrology laboratory for fertility evaluation.

Mesoporous silica nanoparticles inhibit cellular respiration.

We studied the effect of two types of mesoporous silica nanoparticles, MCM-41 and SBA-15, on mitochondrial O 2 consumption (respiration) in HL-60 (myeloid) cells, Jurkat (lymphoid) cells, and isolated mitochondria. SBA-15 inhibited cellular respiration at 25-500 microg/mL; the inhibition was concentration-dependent and time-dependent. The cellular ATP profile paralleled that of respiration.

Caspase activation by anticancer drugs: the caspase storm.

This study measures the time-dependence of cellular caspase activation by anticancer drugs and compares it with that of cellular respiration. Intracellular caspase activation and cellular respiration were measured during continuous exposure of Jurkat, HL-60, and HL-60/MX2 (deficient in topoisomerase-II) cells to dactinomycin, doxorubicin, and the platinum (Pt) compounds cisplatin, carboplatin, and oxaliplatin. Caspase activation was measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC).

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.

Inhibition of cellular respiration by doxorubicin.

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)].

Enhanced cellular respiration in cells exposed to doxorubicin.

Doxorubicin executes topoisomerase II mediated apoptosis, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores (PTP). To further define the effects of doxorubicin on cell metabolism, we measured cellular respiration, cellular ATP, DNA fragmentation, and cytochrome c leakage in Jurkat (supersensitive), human leukemia-60 (HL-60, sensitive), and HL-60/MX2 (resistant) cells following exposure to 1.0 microM doxorubicin for 30 min.

Pi binding by the F1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane.

Pi binding by the F(1)-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane (E. coli F(1)) was examined by two methods: the centrifuge column procedure [Penefsky, H.S.

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.

Immediate effects of anticancer drugs on mitochondrial oxygen consumption.

The evolving role of mitochondria as a target for many anticancer drugs (e.g. platinum-based compounds, alkylating agents and anthracyclines) prompted us to investigate their immediate effects on the mitochondrial respiratory chain.

Reconstitution of beef heart mitochondrial F0F1 in reverse phase evaporation vesicles.

Beef heart mitochondrial F0F1 was reconstituted in proteoliposomes by a new procedure. MF0F1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF0F1-REV). Reconstitution was mediated by Triton X-100, which was subsequently removed by treatment with Bio-Beads.

Energetics of ATP dissociation from the mitochondrial ATPase during oxidative phosphorylation.

The dissociation constant (KdATP) for ATP bound in the high affinity catalytic site of membrane-bound beef heart mitochondrial ATPase (F1) was calculated from the ratio of the rate constants for the reverse dissociation step (k-1) and the forward binding step (k+1). k-1 for ATP bound to submitochondrial particles or to submitochondrial particles washed with KCl so as to activate ATPase activity was accelerated by about five orders of magnitude during respiratory chain-linked oxidations of NADH. In the presence of NADH and 0.

Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart.

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F1 supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP. The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k+1) and the reverse dissociation step (k-1). k-1 was accelerated several orders of magnitude by NADH oxidation.

Covalent modification of catalytic sites on membrane-bound beef heart mitochondrial ATPase by 2-azido-adenine nucleotides.

Incubation in the dark of 32P-labeled 2-azido-adenine nucleotides with submitochondrial particles from beef heart led to tight binding of the label by membrane-bound F1. That is, the label remained with the particles following two passages through centrifuge columns. After removal of free nucleotides and ultraviolet irradiation, the radioactive label was covalently bound exclusively to the beta subunit of the ATPase.

Unisite catalysis and the delta subunit of F1-ATPase in Escherichia coli.

The 5-subunit form of the Escherichia coli F1-ATPase, characterized by the subunit composition alpha 3 beta 3 gamma delta epsilon, failed to exhibit a rate acceleration when samples of the enzyme hydrolyzing substoichiometric concentrations of [gamma-32P]ATP were switched from unisite to multisite hydrolysis by the addition of a cold chase. A 4-subunit form of the enzyme lacking in the delta subunit (alpha 3 beta 3 gamma epsilon) did exhibit cold chase-promoted accelerations in the hydrolysis of ATP. Reconstitution of a 5-subunit enzyme by incubating the 4-subunit form of the enzyme with a purified preparation of subunit delta was accompanied by a disappearance in the response to a cold chase.

Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase.

The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 by maleimides such as N-ethylmaleimide or fluorescein maleimide resulted in a reconstitutively active enzyme that was indistinguishable from the native protein.

The adenine nucleotide translocase modulates oligomycin-induced quenching of pyranine fluorescence in submitochondrial particles.

Incorporation of the fluorescent, nonpermeant pH indicator pyranine into submitochondrial particles (pyranine-SMP) permitted monitoring of intravesicular pH changes brought about by proton translocation due to oxidation of respiratory chain substrates or to hydrolysis of ATP. Addition of oligomycin to beef heart pyranine-SMP was followed by a pH-independent quenching of pyranine fluorescence. Quenching was influenced by the presence of adenine nucleotides both inside and outside the submitochondrial particles.