An assessment of the usefulness of a coconut as a model of the human skull for forensic identification of a homicide weapon.

The authors made an attempt to verify if a coconut can be used as a model of human skull to determine the homicide weapon. During our experiment 27 strike attempts were performed with the use of 9 different tools. Among them there were authentic murder weapons and instruments which had been used in similar experiments conducted on human skulls in 1955. Depending on the size of an area in contact with a coconut, weapons caused dents corresponding to the shape of a weapon, irregular fractures or long linear cracks. Our results have shown that coconut can be used as an inexpensive screening model of human skull, but only to determine fractures made by tools with small striking surface.

Effect of glucose and deoxyglucose on the redistribution of calcium in ehrlich ascites tumour and Zajdela hepatoma cells and its consequences for mitochondrial energetics. Further arguments for the role of Ca(2+) in the mechanism of the crabtree effect.

The distribution of Ca(2+) in intact cells was monitored with fluorescent probes: fura-2 for cytosolic [Ca(2+)] and rhod-2 for mitochondrial [Ca(2+)]. It was found that in neoplastic cells, such as Ehrlich ascites tumour and Zajdela hepatoma, but not in non-malignant cells, such as fibroblasts, glucose and deoxyglucose elicited release of Ca(2+) from endoplasmic reticulum stores and an increase in Ca(2+) concentration in the cytosol. Parallel to this, a decrease in the rate of Ca(2+) extrusion from the cell and an enhanced uptake of Ca(2+) by mitochondria were observed. The increase in mitochondrial [Ca(2+)] was accompanied by an increase in the mitochondrial membrane potential and the reduction state of nicotinamide nucleotides. F(1)F(o)-ATPase in submitochondrial particles of Zajdela hepatoma was strongly inhibited in the presence of micromolar Ca(2+) concentrations, whereas this activity in submitochondrial particles from rat liver appeared to be less sensitive to Ca(2+). Indications of glycosylation of Ehrlich ascites tumour cell proteins were also obtained. These data strengthen the proposal [Bogucka, K., Teplova, V.V., Wojtczak, L. and Evtodienko, Y. V. (1995) Biochim. Biophys. Acta 1228, 261-266] that the Crabtree effect is produced by mobilization of cell calcium, which is subsequently taken up by mitochondria and inhibits F(1)F(o)-ATP synthase.

Inhibition by Ca2+ of the hydrolysis and the synthesis of ATP in Ehrlich ascites tumour mitochondria: relation to the Crabtree effect.

Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumour cells is greatly reduced when the mitochondria have been preloaded with Ca2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca(2+)-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca2+, indicating that, independently of adenine nucleotide transport, F1Fo-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F1Fo-ATPase (the Pullman-Monroy protein inhibitor) is insensitive to Ca2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca2+ (Teplova, V.V., Bogucka, K., Czyz, A., Evtodienko, Yu.V., Duszynski, J. and Wojtczak, L. (1993) Biochem. Biophys. Res. Commun. 196, 1148-1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V., Teplova, V.V., Duszynski, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439-446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.

The role of cytoplasmic [Ca2+] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect.

The effect of Ca2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (delta psi) and alkalinization of the medium. This typical behaviour is drastically changed if Ca2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of delta psi, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 microM Ca2+ and a preincubation for 2 min with 0.4-1.0 microM Ca2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca2+ concentrations as low as 0.3 microM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyz A. Evtodienko YuV. Duszynski J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148-1154; Czyz A. Teplova VV. Sabala P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539-544], the present results suggest that cytoplasmic Ca2+ plays a crucial role in the Crabtree effect.

Effect of glucose and deoxyglucose on cytoplasmic [Ca2+] in Ehrlich ascites tumor cells.

Concentration of free cytoplasmic Ca2+ ([Ca2+]i) in Ehrlich ascites tumor cells, measured using fura-2, amounted to 170-300 nM and was increased by 50-160 nM after addition of 10 mM D-glucose or D-2-deoxyglucose but not 3-O-methylglucose at pH 7.4. In the range of external pH between 6.8 and 7.8 the increase was higher at higher pH. This increase occurred within 30-60 s after addition of hexose and lasted for at least 10 min. This [Ca2+]i rise was observed both in presence and virtual absence of Ca2+ in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+] in the external medium. Pretreatment of the cells with thapsigargin resulted in a much smaller [Ca2+]i increase after addition of glucose or deoxyglucose. The mechanism of [Ca2+]i rise evoked by glucose and deoxyglucose and its importance in switching cell metabolism from oxidative to glycolytic are discussed.

Regulation of mitochondrial resting state respiration: slip, leak, heterogeneity?

The contribution of molecular slippage of proton pumps, of proton leak and of coupling heterogeneity of mitochondrial population to the well-known non-linear interrelationship between resting state respiration and the protonmotive force is discussed in view of the following experimental findings. (1) After blocking mitochondrial respiration with cyanide, the rate of dissipation of the membrane potential is non-linearly dependent on the actual membrane potential, similarly to the resting state respiration in mitochondria titrated with small amounts of an inhibitor. In contrast, delta pH dissipates proportionally to its actual value. (2) The rate of electron flow from succinate to ferricyanide depends upon the protonmotive force, similarly to the flow from succinate to oxygen. This strongly suggests that the H+/e- stoichiometry in complexes III and IV of the respiratory chain is constant. (3) Mitochondria 'in situ', in permeabilized Ehrlich ascites cells, exhibit the same non-linear flux/force relationship as isolated mitochondria. These results strongly suggest that the non-ohmic characteristics of the inner mitochondrial membrane, with respect to protons driven by the membrane potential but not by the concentration gradient, is the main factor responsible for the nonlinear flux/force relationship in resting state mitochondria.

Energetics of Ehrlich ascites mitochondria: membrane potential of isolated mitochondria and mitochondria within digitonin-permeabilized cells.

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.

Homeostasis of the protonmotive force in phosphorylating mitochondria.

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.

Relationship between the energy cost of ATP transport and ATP synthesis in mitochondria.

Liver mitochondria form rats kept on a high-protein diet exhibit an increased rate of respiration upon addition of ornithine in the presence of HCO2 and NH+4. This is the manifestation of intramitochondrial utilization of ATP for the synthesis of citrulline. State 3 respiration of these mitochondria could be adjusted to the same rate as that produced by ornithine by either using limiting amounts of hexokinase or titration with atractyloside. Under such conditions, in both systems the proton-motive force, the intramitochondrial ATP/ADP ratio and the redox state of the respiratory chain were the same. In contrast to this, the ATP/O ratio (equal to 2 X citrulline/O ratio) in mitochondria synthesizing citrulline was higher than the glucose 6-phosphate/O ratio in the system where ATP was trapped extramitochondrially. The ratio of these two ratios was close to 1.5 with both glutamate and succinate as respiratory substrates. From these results it can be concluded that the translocation of ATP against ADP and phosphate utilizes an amount of the chemiosmotic proton gradient equal to half of that needed for the synthesis of ATP in the inner compartment.

Influence of different energy drains on the interrelationship between the rate of respiration, proton-motive force and adenine nucleotide patterns in isolated mitochondria.

The respiration of rat liver mitochondria was stimulated by three different ways of energy drain: (a) partial uncoupling (equivalent to direct collapse of the proton-motive force), (b) intramitochondrial utilization of ATP for citrulline synthesis, and (c) extramitochondrial utilization of ATP for glucose phosphorylation. At identical rates of respiration, the intramitochondrial ATP : ADP ratios were the same in all three systems. Furthermore, the proton-motive force was the same in partially uncoupled mitochondria and in the presence of hexokinase plus glucose up to a respiration rate amounting to about 60% of that of the fully active state. However, external ATP : ADP ratios were considerably different in various systems at comparable rates of oxygen uptake, being the lowest under conditions when ATP was being utilized externally. On this basis, it is concluded that the respiratory rate is controlled directly by the proton-motive force and the mitochondrial ATP-synthesizing system operates under near-equilibrium conditions with respect to the membrane energy state parameters. However, a disequilibrium exists at the step of the transport of ATP from mitochondria to the external (cytoplasmic) compartment.