Measurement of mitochondrial transmembrane electric potential using the fluorescent probe DSM]

The measurement of transmembrane electric potential of rat liver mitochondria was performed with fluorescent probe DSM. In the incubation medium with succinate the electric potential was -190 +/- 5 mV. Complete uncoupling caused by CCCP in the concentration 0.5 microM decreased the potential to -142 +/- 3 mV. In the partial uncoupling with a half of the mentioned CCCP concentration the potential was -158 +/- 6 mV. DSM influence on oxidation and phosphorylation was not detected.     

Effect of ethanol on accumulation of Ca ions in intracellular structures of rat myometrium under conditions of first creating ethanol-dependent models

In the experiments which have been conducted on digitonin-treated myometrium cell suspensions of nonpregnant rats the direct influence of ethanol in concentration 0-10% on the Ca2+ accumulation in mitochondria and endoplasmic reticulum have been studied. Studies have been conducted on the different models, such as, control (model I), subacute (model II), acute (model III) and chronic ethanol consumption (model IV). It has been shown for all models that the dependence of Ca2+ accumulation by mitochondria on the concentration of ethanol in incubation medium is bell-shaped. Acute and chronic ethanol consumption resulted in statistically reliable decrease in the amount of accumulated cations. Nevertheless the I50 values were the same for the models I-III and were 8-9%, although in the case of model IV this one was only 4.0 +/- 0.6%. The increase of ethanol concentration in the incubation medium caused of Ca2+ accumulation decreasing in the endoplasmic reticulum for all studied models, the values of I50 also decreased for models II-IV (2.8 +/- 0.2; 2.5 +/- 0.2 and 2.3 +/- 0.3% respectively) relative to the control (3.8 +/- 0.2%). At the level of model I oxytocin-inhibited component of the endoplasmic reticulum Ca2+ uptake was more stable to the ethanol effects than oxytocin-independent one. Although the sensitivity of the first one to the ethanol effects at the level of models II-IV rose, that parameter for the oxytocin-independent component was not changed. The mechanisms of ethanol effects on Ca2+ accumulation in the myometrium intracellular structures have been discussed.     

Ionic strength of the intermembrane space of intact mitochondria as estimated with fluorescein-BSA delivered by low pH fusion

The electrostatic interactions of cytochrome c with its redox partners and membrane lipids, as well as other protein interactions and biochemical reactions, may be modulated by the ionic strength of the intermembrane space of the mitochondrion. FITC-BSA was used to determine the relative value of the mitochondrial intermembrane ionic strength with respect to bulk medium external to the mitochondrial outer membrane. FITC-BSA exhibited an ionic strength-dependent fluorescence change with an affinity in the mM range as opposed to its pH sensitivity in the microM range. A controlled, low pH-induced membrane fusion procedure was developed to transfer FITC-BSA encapsulated in asolectin liposomes, to the intermembrane space of intact mitochondria. The fusion procedure did not significantly affect mitochondrial ultrastructure, electron transport, or respiratory control ratios. The extent of fusion of liposomes with the mitochondrial outer membrane was monitored by fluorescence dequenching assays using a membrane fluorescent probe (octadecylrhodamine B) and the soluble FITC-BSA fluorescent probe, which report membrane and contents mixing, respectively. Assays were consistent with a rapid, low pH-induced vesicle-outer membrane fusion and delivery of FITC-BSA into the intermembrane space. Similar affinities for the ionic strength-dependent change in fluorescence were found for bulk medium, soluble (9.8 +/- 0.8 mM) and intermembrane space-entrapped FITC-BSA (10.2 +/- 0.6 mM). FITC-BSA consistently reported an ionic strength in the intermembrane space of the functionally and structurally intact mitochondria within +/- 20% of the external bulk solution. These findings reveal that the intermembrane ionic strength changes as does the external ionic strength and suggest that cytochrome c interactions, as well as other protein interactions and biochemical reactions, proceed in the intermembrane space of mitochondria in the intact cell at physiological ionic strength, i.e., 100-150 mM.     

Nitric oxide production and mitochondrial dysfunction during rat thymocyte apoptosis

Production of nitric oxide (NO) by mitochondrial membranes as methemoglobin formation sensitive to N(G)-methyl-l-arginine inhibition and mitochondrial O(2) consumption in metabolic states 3 and 4 and the respiratory control (state 3/state 4) were measured at early stages of rat thymocyte apoptosis. Programmed cell death was induced by addition of methylprednisolone and etoposide to thymocyte suspensions. Increased NO production by mitochondrial membranes was observed after 30 min of methylprednisolone and etoposide addition and was accompanied by mitochondrial respiratory impairment as an early phenomenon in apoptotic thymocytes. The respiratory control in isolated mitochondria from untreated thymocytes was 4.2 +/- 0.2 and decreased to 3.1 +/- 0.2 and 1.9 +/- 0.3 after 1 h of methylprednisolone and etoposide treatment, respectively. The low mitochondrial respiratory control was accompanied by a marked decrease in GSH and cytochrome c content. Moreover, an inhibitory effect in the amount of apoptosis due to thymocyte pretreatment with N(G)-methyl-l-arginine and N(omega)-nitro-(l)-arginine (l-NNA), indicate that nitric oxide production is closely involved in the signaling of rat thymocyte apoptosis.     

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

A method is presented for the preparation of pure phthalonic acid (PTA) in high yields. This PTA was used to determine the capacity of the malate/aspartate shuttle in pea (Pisum sativum) leaf mitochondria. The inhibition of glycine-dependent O2 uptake in the combined presence of 5 mM-aspartate and 5 mM-2-oxoglutarate (2-OG) was decreased by 55 +/- 22% (n = 13) in washed and 50 +/- 2% (n = 11) in purified mitochondria by 0.23 mM-PTA. This concentration of PTA had no effect on the oxidation of 5 mM-2-OG, suggesting that part of the observed inhibition of O2 uptake in the presence of aspartate and 2-OG was due to the production of oxaloacetate (OAA) by aspartate aminotransferase external to the mitochondrial inner membrane. Levels of external aspartate aminotransferase were estimated to be 24 +/- 1% (n = 4) and 13 +/- 1% (n = 4) of the total mitochondrial activity in washed and purified mitochondria respectively. Malate/aspartate-shuttle activity was estimated directly by measuring rates of malate efflux from isolated mitochondria and was found to match estimates of shuttle activity based on the PTA-insensitive inhibition of O2 uptake. Comparisons of malate/aspartate- and malate/OAA-shuttle activities indicated potentially similar rates of NADH export from pea leaf mitochondria under conditions in vivo. These extrapolated to whole-tissue rates of 5-11 mumol of NADH.h-1.mg of chlorophyll-1. The potential role of the malate/aspartate shuttle in the support of photorespiratory glycine oxidation in leaf tissue is discussed.     

Anthracycline binding to synthetic and natural membranes. A study using resonance energy transfer

The binding of adriamycin and its two analogues 4'-epidoxorubicin and 4'-deoxydoxorubicin to synthetic and mitochondrial membranes was investigated by using resonance energy transfer between these drugs and two fluorescent probes, diphenylhexatriene (DPH) and tryptophan. The fluorescence of the lipid probe DPH in both types of membranes and tryptophan in mitochondria was quenched by the anthracyclines in a dose-dependent manner. In sonicated, fluid-phase dimyristoyl-L-alpha-phosphatidylcholine (DMPC) vesicles, the half-quenching concentration (K50) of adriamycin was 17 +/- 1 microM, whereas in bilayers containing a 1:1 molar ratio of DMPC to cardiolipin (CL), the value was 8 +/- 1 microM. In liver and heart mitochondria, the K50 values were 8 +/- 2 and 11 +/- 3 microM, respectively. Similar results were obtained for the other two drugs. Replacing a nonionic with an ionic medium or decreasing the pH from pH 7.7 to pH 6.9 increased the K50 value of adriamycin for DPH in DMPC/CL (1:1 molar) liposomes and in mitochondria. Higher concentrations of anthracycline were needed to quench the fluorescence of tryptophan. The results suggest that these drugs interact with both phospholipids and proteins and that the cardiotoxicity of adriamycin is unlikely to be related to the amount of drug bound to heart mitochondria.     

Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter

A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.     

Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells

A new biochemical method for estimating the virtual number of mitochondria (mt) per cell was developed and used together with a plasmid probe to measure mt DNA/mitochondrion and mt DNA/cell. These methods were used in five cell types from four mammalian species. Mt DNA/mitochondrion was essentially constant in all cell types (mean 2.6 +/- 0.30 SE mitochondrial DNA molecules/mt). Mt DNA molecules/cell encompassed an eight-fold range between various cell types (low 220 +/- 6.2; high 1,720 +/- 162 mt DNA molecules/cell). Virtual mt number/cell ranged from 83 +/- 17 to 677 +/- 80 (SE) mt/cell in various cell types. All five mammalian virtual mitochondria contained the same genomic mass. The number of virtual mitochondria per cell and amount of mt DNA per cell appear to be closely regulated within a given cell type but differ widely from cell type to cell type.     

Mitochondrial membrane potential estimated with the correction of probe binding

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and/or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 microM) and low (under 20 microM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25 degrees C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 microM.     

Functional relationship between the ADP/ATP-carrier and the F1-ATPase in mitochondria.

1. The distribution of labeled and unlabeled adenine-nucleotides inside and outside mitochondria was followed after addition of [14C]ADP to rat liver mitochondria. Two types of mitochondria were used: 1, respiring mitochondria which were carrying out oxidative phosphorylation and which had been replenished in ATP by incubation in a medium supplemented with succinate and phosphate; 2, non-respiring mitochondria which had been partially depleted of ATP by incubation in a medium supplemented with rotenone and phosphate. During the first minute following addition of [14C]ADP to the respiring mitochondria, the pre-existing intramitochondrial (internal) [12C]ATP was released into the medium and replaced by newly synthesized [14C]ATP. No [14C]ADP accumulated in the mitochondria. It is suggested that extramitochondrial (external) ADP entering respiring mitochondria in exchange for internal ATP is phosphorylated to ATP before its complete release in the matrix space. In non-respiring mitochondria, the entry of [14C]ADP into the mitochondria was accompanied by the appearance in the external space of [12C]ADP and [12C]ATP, with a marked predominance of [12C]ADP. Thus in non-respiring mitochondria, the residual internal ATP is dephosphorylated to ADP in the inner membrane before being released outside the mitochondria. 2. When mitochondria were incubated with glutamate, ADP and [32P]phosphate, the [32P]ATP which accumulated in the matrix space became rapidly labeled in both the P gamma and P beta groups of the ATP, due to the presence of a transphosphorylation system in the mitochondrial matrix. The [32P]ATP which accumulated outside the mitochondria was also labeled in the P beta group, although less rapidly than the internal ATP. Our data show that a large fraction (75-80%) of the ATP produced by phosphorylation of added ADP within the inner mitochondrial membrane is released into the matrix space before being transported out from the mitochondria; only a small part (20-25%) is released directly outside the mitochondria without penetrating the matrix space. 3. In respiring and phosphorylating mitochondria, the value of the Km of the ADP-carrier for external ADP was 2-4 times lower than its value in non-respiring and non-phosphorylating mitochondria. 4. The above experimental data are discussed with reference to the topological and functional relationships between the ADP-carrier and the oxidative phosphorylation complex in the inner mitochondrial membrane. They strongly suggest that the ADP-carrier comes to the close neighbourhood of the ATP synthetase on the matrix side of the inner membrane.     

Stimulating effect of erythropoietin on thymocyte energetics established in vitro with a potential-sensitive fluorescent probe

Effects of a peptide hormone—human recombinant erythropoietin (EPO)—on transmembrane potential (TMP) and a number of active mitochondria in rat thymocytes were studied in vitro using the fluorescent cationic probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). It was established that EPO changes electric potentials on the surface of cellular membranes of rat thymocytes. Changes in fluorescent signals of DSM were found to depend on the EPO concentration and physiological status of thymus cells. EPO concentrations sufficient to increase average fluorescence intensity of DSM ( $\tilde F$ ) in thymus cell mitochondria were established in vitro. These effects were stimulated by increasing the average number of energized mitochondria (Ñ _m ) able to accumulate the fluorescent cationic probe. These changes can also be due to elevation of proton potential on the mitochondrial membrane and/or of electric potential of the plasma membrane. Experimental values of positive correlation coefficients between mean values of $\tilde F$ and Ñ _m in experimental (EPO) thymus cell samples differed from those in control. In the presence of EPO, $\tilde F$ increased nonlinearly with Ñ _m due to different responsiveness of thymocytes to EPO and change in polarization of the outer mitochondrial membrane in some EPO-stimulated cells. In the majority of cases, the peak in the distribution histogram of $\tilde F$ was shifted towards augmentation of the DSM signal. The EPO response of cells isolated from different thymuses depended on the initial level of the average fluorescent signal of DSM in mitochondria, which testifies to differences in physiological statuses of animal thymuses and experimental animals at large.     

Primary role of sarcoplasmic reticulum in phasic contractile activation of cardiac myocytes with shunted myolemma

Homogeneous populations of single myocytes showing good preservation of ultrastructure were obtained by enzymatic digestion of rabbit and rat hearts, and maintained in a relaxed state in the presence of free Ca2+ concentrations less than 10(-7) M. Ultrastructural details such as a cytoskeleton of 100-A filaments connected to the sarcolemma at the Z lines were demonstrated especially well in these preparations. In spite of seemingly normal structure, electron probe analysis of cryosections reveals similar concentrations of electrolytes in the medium and in the cytoplasm, indicating the presence of electrochemical shunting across the external membrane. The dissociated myocytes display Ca uptake and phasic contractions that are apparently dependent on mitochondrial respiration, but are not affected by mitochondrial uncouplers when ATP and phosphocreatine are added. The uptake is augmented by oxalate and, based on identification of calcium oxalate crystals by electron microscopy and electron probe analysis, is localized to the sarcoplasmic reticulum (SR). An advantageous feature of the dissociated myocytes is that they are suitable for experiments using large numbers of cells in suspension. Thereby, velocities of calcium transport were measured directly by isotopic tracer and filtration methods. It was then found that the lowest CA2+ concentrations (5 x 10(-7) M for the rabbit and 1 x 10(-7) M for the rat) sustaining Ca transport also induce phasic contractile activity in all myocytes, even though the external membrane is electrochemically shunted. A stepwise rise in the Ca2+ concentration of up to one order of magnitude, increases transport velocities in parallel with the rates of phasic contractions. Both these parameters are affected by Mg2+, temperature, cyclic-AMP, and methylxanthines, even though the Ca2+ concentration is maintained constant in the medium. Therefore, Ca transport by SR is a requirement and a rate limiting factor for the occurrence of phasic contractile activation in dissociated cardiac cells retaining an electrochemically shunted external membrane. It is suggested that transient Ca release required for phasic contractile activation is due to equilibrium oscillations across the SR membrane. The sequential pattern of sarcomere activation is consistent with a self propagating mechanism of calcium release. SR in dissociated skeletal muscle cells sustains a greater Ca transport activity than in dissociated heart cells. However, the heart cells display a much higher phasic contractile activity, indicating that cardiac SR has a greater tendency to release accumulated calcium. If free Ca2+ in the medium is raised above 10(-6) M, both cardiac and skeletal myocytes undergo contractures and degenerative phenomena, accompanied by Ca, Mg, and phosphate accumulation in cardiac mitochondria.     

The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria

Characteristics of the succinate-supported H(2)O(2) formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H(2)O(2) generation measured in mitochondria prepared with digitonin (600.6+/-26.8pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H(2)O(2) formation was significantly smaller in Percoll-purified mitochondria (252.6+/-17.3pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H(2)O(2) formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185+/-3.2mV) and addition of BSA (0.025%) to the assay medium increased H(2)O(2) generation by only 50%. In Percoll-purified mitochondria DeltaPsim was more depolarized (171+/-2mV) and BSA caused hyperpolarization by 10.7+/-1.9mV. H(2)O(2) formation, which was largely independent of DeltaPsim, was stimulated by 400%, became highly dependent on DeltaPsim and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on DeltaPsim.     

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria

Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).     

The internal concentration of K+ in isolated rat liver mitochondria

A simple osmotic method has been developed to determine the internal K+ concentration of mitochondria by determining the concentration of external K+ at constant osmotic pressure at which metabolically inhibited mitochondria neither shrink nor swell. This concentration has been found to correspond to approx. 80-85 mM in freshly isolated mitochondria and considerably lower after additional centrifugation procedures. Since mitochondria are in osmotic equilibrium with the suspending medium (in this case, 0.32 osmolal), and K+ is the primary exchangeable internal ion, a significant proportion of the internal osmotic pressure must be exerted by the sucrose. Results for experiments determining internal K+ after centrifuging mitochondria at various G values confirm the reports of Sitaramam et al. (Sitaraman, V. and Sarma, M.K.J. (1981) Proc. Natl. Acad. Sci. USA 78, 3441-3445 and Sambasivarao, D. and Sitaramam, V. (1983) Biochim. Biophys. Acta 722, 256-270) that centrifugation induces the entry of sucrose in mitochondria isolated in a sucrose medium.     

Increasing the concentration of free calcium in thymocytes during gamma-irradiation may induce their death

Free Ca2+ concentration in thymocytes increased 0.5-1.5h after gamma-irradiation (10 Gy) as was measured by Quin-2AM fluorescent probe. Cycloheximide, a protein synthesis inhibitor, suppressed Ca2+ increase and inhibited radiation-induced thymocyte death. EL-4 thymoma cells did not exhibit any changes in free Ca2+ concentration and interphase death after gamma-irradiation. It is believed that the radiation-induced increase of free Ca2+ concentration in thymocytes may induce their death.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Involvement of sodium in early phosphatidylserine exposure and phospholipid scrambling induced by P2X7 purinoceptor activation in thymocytes

Extracellular ATP (ATP(ec)), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP(ec) induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP(ec)-induced Ca(2+) and/or Na(+) influx. At physiological external Na(+) concentration, without external Ca(2+), PS was exposed in all ATP(ec)-responsive cells. In contrast, without external Na(+), physiological external Ca(2+) concentration promoted a submaximal response. Altogether these data show that Na(+) influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.     

Human thymic epithelial cells function as accessory cells for autologous mature thymocyte activation

Using human thymocytes and autologous thymic epithelial (TE) cells grown in vitro in long-term culture, we have found TE cells can function as accessory cells for mitogen-induced mature thymocyte activation. Tritiated thymidine incorporation, blast formation, and protein synthesis were all induced in accessory cell-depleted thymocytes by autologous TE cells in the presence of suboptimal concentrations of PHA. After 3 days of mitogen stimulation of thymocyte-TE cell cocultures in vitro, thymocyte blasts bound to TE cells and 77 +/- 4% (mean +/- SEM) of TE cells acquired expression of major histocompatibility complex (MHC) class II (DR) antigen. TE accessory cell function for thymocyte activation was dependent on the number of TE cells added to thymocyte cultures, was not dependent on TE cell division, but did require TE cell protein synthesis. In thymocyte separation experiments, the predominant cell type responding to PHA in the presence of TE cells was T6- mature (stage III) thymocytes. Thus, human TE cells are capable of providing signals that lead to mature thymocyte activation.     

Electrical Properties of Mitochondrial Membranes

The electrical capacity of the membrane of rat liver mitochondria is 0.5 to 0.6 µ./cm². This membrane capacity is obtained from the analysis of the frequency dependence of the admittance of a suspension of swollen mitochondria. In potassium chloride media the mitochondrial membrane capacity does not depend on the ion concentration. The internal conductance of the mitochondria was approximately one-half that of the external medium; the same applies if the mitochondria are equilibrated in a medium with a 10-fold difference in potassium chloride concentration. Hence the swollen mitochondria investigated here appear to be able to adjust their internal ion concentration in proportion with that of the external phase. The similarity of the membrane capacity of isolated mitochondria with the range of values known for other membranes suggests a common molecular structure. The analysis of experimental data suggests an anisotropic electrical behavior of the interior of mitochondria. This anisotropy is readily explained by the existence of internal membranes.