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Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and 'ghost' fibers (all with a diameter of 10-20 µm) at different (0-3 µmol 1[sup -1]) free Ca[sup 2+] concentrations in the medium. In all these preparations, the apparent K[sub m] of mitochondrial respiration for exogenous ADP at free Ca[sup 2+] concentrations of 0-0.1 µmol l[sup -1] was very high, in the range of 250-350 µmol 1[sup -1], in contrast to isolated mitochondria in vitro (apparent K[sub m] for ADP is approximately 20 µmol l[sup -1]). An increase in the free Ca[sup 2+] concentration (up to 3 µmol l[sup -1], which is within physiological range), resulted in a very significant decrease of the apparent K[sub m] value to 20-30 µmol l[sup -1], a decrease of V[sub max] of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent K[sub m] for ADP (300-350 µmol 1[sup -1]) nor V[sub max] of respiration changed in the range of free Ca[sup 2+] concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryltransfer networks, and apparently depends on the state of sarcomere structures. [ABSTRACT FROM AUTHOR]

The functional role of platelets is intricately linked to the dynamic organization of two main components of the cytoskeleton, microtubules and actin fibers. Throughout the phases of platelet activation, spreading, and retraction, both of these essential polymers undergo continuous and orchestrated reorganization. Our investigation of the dynamic cytoskeletal changes during these phases highlights a sequential remodeling of the actin cytoskeleton in adherent platelets from the formation of initial actin nodules through the development of stress fibers and a subsequent return to nodular structures. Concurrently, the marginal ring of microtubules, characteristic of resting platelets, undergoes a re-organization induced by marginal band extension and coiling toward the formation of star-like bundles of microtubules. Subsequently, these bundles are dispersed into individual microtubules, which are re-bundled at later stages before ring-like structures are formed again. These findings suggest a compelling tendency for both cytoskeletal components to revert to their original configurations. Notably, the early steps of platelet cytoskeleton reorganizations have previously been shown to be regulated by the signaling cascade triggered during platelet activation, which leads to an increase of cytosolic calcium concentrations. We show here that later steps are potentially regulated by a progressive decrease of intracellular calcium concentrations as platelets approach the end of their functional lifespan.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Motor skill learning requires the activity of the dorsal striatum, with a differential global implication of the dorsomedial and dorsolateral territories. We investigate here whether and how specific striatal neurons encode the acquisition and consolidation of a motor skill. Using ex vivo two-photon calcium imaging after rotarod training, we report that highly active (HA) striatal populations arise from distinct spatiotemporal reorganization in the dorsomedial (DMS) and dorsolateral (DLS) striatum networks and are correlated with learning performance. The DMS overall activity decreases in early training, with few and sparsely distributed HA cells, while the DLS shows a progressive and long-lasting formation of HA cell clusters. These reorganizations result from reinforcement of synaptic connections to the DMS and anatomical rearrangements to the DLS. Targeted silencing of DMS or DLS HA cells with the cFos-TRAP strategy strongly impairs individual performance. Our data reveal that discrete domains of striatal populations encode acquisition and long-lasting retention of a motor skill., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neurodevelopmental axonal pathfinding plays a central role in correct brain wiring and subsequent cognitive abilities. Within the growth cone, various intracellular effectors transduce axonal guidance signals by remodeling the cytoskeleton. Semaphorin-3E (Sema3E) is a guidance cue implicated in development of the fornix, a neuronal tract connecting the hippocampus to the hypothalamus. Microtubule-associated protein 6 (MAP6) has been shown to be involved in the Sema3E growth-promoting signaling pathway. In this study, we identified the collapsin response mediator protein 4 (CRMP4) as a MAP6 partner and a crucial effector in Sema3E growth-promoting activity. CRMP4-KO mice displayed abnormal fornix development reminiscent of that observed in Sema3E-KO mice. CRMP4 was shown to interact with the Sema3E tripartite receptor complex within detergent- resistant membrane (DRM) domains, and DRM domain integrity was required to transduce Sema3E signaling through the Akt/GSK3 pathway. Finally, we showed that the cytoskeleton-binding domain of CRMP4 is required for Sema3E's growth-promoting activity, suggesting that CRMP4 plays a role at the interface between Sema3E receptors, located in DRM domains, and the cytoskeleton network. As the fornix is affected in many psychiatric diseases, such as schizophrenia, our results provide new insights to better understand the neurodevelopmental components of these diseases., Competing Interests: BB, CR, AP, CB, CD, FA, ED, AK, MJ, AF, AA, SG, JD No competing interests declared, (© 2021, Boulan et al.)

The synthesis of keto-heptamethine derivatives has been expanded to various new symmetrical and asymmetrical structures, including an unprecedented di-anionic keto-polymethine. The spectroscopic behavior of these new dyes has been systematically and thoroughly investigated, revealing that the formation of hydrogen bond interactions with protic solvents is responsible for a dramatic enhancement of the fluorescence quantum yield in the far-red spectral region. The existence of these strong hydrogen-bond interactions was further confirmed by molecular dynamics simulations. These bis-dipolar polymethines exhibit large two-photon absorption (TPA) cross-sections ( σ 2 in GM) in the near-infrared, making them ideal candidates for NIR-to-NIR two-photon microscopy imaging applications. We demonstrate that the molecular engineering of the hydrophilic/hydrophobic balance enables targeting of different cellular components, such as cytoplasm or cell membranes. Addition of appropriate substituents provides the molecule with high-water-solubility, affording efficient two-photon probes for angiography.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80 μm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D ⊥ ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a - 28% decrease in FA (p < 0.005) and a + 95% increase in D ⊥ (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D ⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D ⊥ as biomarkers for glioma cell invasion., (Copyright © 2016 John Wiley & Sons, Ltd.)

A tris-cyclometalated iridium complex that bears two ligands functionalized by peripheral carbazole groups combines an intense solid state emission and a significant two-photon absorption cross section in the near-infrared. After incorporation into a physiological micellar suspension, it can be used for the intravital two-photon fluorescence microscopy of cerebral vasculature.

Nanoparticles containing high-Z elements are known to boost the efficacy of radiation therapy. Gadolinium (Gd) is particularly attractive because this element is also a positive contrast agent for MRI, which allows for the simultaneous use of imaging to guide the irradiation and to delineate the tumor. In this study, we used the Gd-based nanoparticles, AGuIX®. After intravenous injection into animals bearing B16F10 tumors, some nanoparticles remained inside the tumor cells for more than 24 hours, indicating that a single administration of nanoparticles might be sufficient for several irradiations. Combining AGuIX® with radiation therapy increases tumor cell death, and improves the life spans of animals bearing multiple brain melanoma metastases. These results provide preclinical proof-of-concept for a phase I clinical trial.

Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature.

The storage and catabolism of Ultrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles were analyzed through a multiscale approach combining Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM) at different times after intravenous injection in an atherosclerotic ApoE(-/-) mouse model. The atherosclerotic plaque features and the USPIO heterogeneous biodistribution were revealed down from organ's scale to subcellular level. The biotransformation of the nanoparticle iron oxide (maghemite) core into ferritin, the non-toxic form of iron storage, was demonstrated for the first time ex vivo in atherosclerotic plaques as well as in spleen, the iron storage organ. These results rely on an innovative spatial and structural investigation of USPIO's catabolism in cellular phagolysosomes. This study showed that these nanoparticles were stored as non-toxic iron compounds: maghemite oxide or ferritin, which is promising for MRI detection of atherosclerotic plaques in clinics using these USPIOs. From the Clinical Editor: Advance in nanotechnology has brought new contrast agents for clinical imaging. In this article, the authors investigated the use and biotransformation of Ultrasmall Super-paramagnetic Iron Oxide (USPIO) nanoparticles for analysis of atherosclerotic plagues in Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM). The biophysical data generated from this study could enable the possible use of these nanoparticles for the benefits of clinical patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Despite important human and financial resources and considerable accumulation of scientific publications, patents, and clinical trials, cancer research has been slow in achieving a therapeutic revolution similar to the one that occurred in the last century for infectious diseases. It has been proposed that science proceeds not only by accumulating data but also through paradigm shifts. Here, we propose to use the concept of 'paradigm shift' as a method of investigation when dominant paradigms fail to achieve their promises. The first step in using the 'paradigm shift' method in cancer research requires identifying its founding paradigms. In this review, two of these founding paradigms will be discussed: (i) the reification of cancer as a tumour mass and (ii) the translation of the concepts issued from infectious disease in cancer research. We show how these founding paradigms can generate biases that lead to over-diagnosis and over-treatment and also hamper the development of curative cancer therapies. We apply the 'paradigm shift' method to produce perspective reversals consistent with current experimental evidence. The 'paradigm shift' method enlightens the existence of a tumour physiologic-prophylactic-pathologic continuum. It integrates the target/antitarget concept and that cancer is also an extracellular disease. The 'paradigm shift' method has immediate implications for cancer prevention and therapy. It could be a general method of investigation for other diseases awaiting therapy.

We previously reported the synthesis of gadolinium-based nanoparticles (NPs) denoted AGuIX (activation and guiding of irradiation by X-ray) NPs and demonstrated their potential as an MRI contrast agent and their efficacy as radiosensitizing particles during X-ray cancer treatment. Here we focus on the elimination kinetics of AGuIX NPs from the subcellular to whole-organ scale using original and complementary methods such as laser-induced breakdown spectroscopy (LIBS), intravital two-photon microscopy, inductively coupled plasma optical emission spectrometry (ICP-OES), transmission electron microscopy (TEM), and electrospray ionization mass spectrometry (ESI-MS). This combination of techniques allows the exact mechanism of AGuIX NPs elimination to be elucidated, including their retention in proximal tubules and their excretion as degraded or native NPs. Finally, we demonstrated that systemic AGuIX NP administration induced moderate and transient effects on renal function. These results provide useful and promising preclinical information concerning the safety of theranostic AGuIX NPs.

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214 nm above 37 °C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13 min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Multipotent mesenchymal stromal cells (MSC), have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation. The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair. However, some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist. In brain, perivascular MSCs like pericytes and adventitial cells, could constitute another stem cell population distinct to the neural stem cell pool. The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes, the demonstration of neural biomarkers expression, electrophysiological recordings, and the absence of cell fusion. The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells. It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.

Viewing tumors as ecosystems offers the opportunity to consider how ecological concepts can be translated to novel therapeutic perspectives. The ecological trap concept emerged approximately half a century ago when it was observed that animals can prefer an environment of low quality for survival over other available environments of higher quality. The presence of such a trap can drive a local population to extinction. The cancer cell trap concept is the translation of the ecological trap into glioma therapy. It exploits and diverts the invasive potential of glioma cells by guiding their migration towards specific locations where a local therapy can be delivered efficiently. This illustrates how an ecological concept can change therapeutic obstacles into therapeutic tools.

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.

Background: The substantia nigra pars reticulata (SNr) is a major output nucleus of the basal ganglia, delivering inhibitory efferents to the relay nuclei of the thalamus. Pathological hyperactivity of SNr neurons is known to be responsible for some motor disorders e.g. in Parkinson's disease. One way to restore this pathological activity is to electrically stimulate one of the SNr input, the excitatory subthalamic nucleus (STN), which has emerged as an effective treatment for parkinsonian patients. The neuronal network and signal processing of the basal ganglia are well known but, paradoxically, the role of astrocytes in the regulation of SNr activity has never been studied., Principal Findings: In this work, we developed a rat brain slice model to study the influence of spontaneous and induced excitability of afferent nuclei on SNr astrocytes calcium activity. Astrocytes represent the main cellular population in the SNr and display spontaneous calcium activities in basal conditions. Half of this activity is autonomous (i.e. independent of synaptic activity) while the other half is dependent on spontaneous glutamate and GABA release, probably controlled by the pace-maker activity of the pallido-nigral and subthalamo-nigral loops. Modification of the activity of the loops by STN electrical stimulation disrupted this astrocytic calcium excitability through an increase of glutamate and GABA releases. Astrocytic AMPA, mGlu and GABA(A) receptors were involved in this effect., Significance: Astrocytes are now viewed as active components of neural networks but their role depends on the brain structure concerned. In the SNr, evoked activity prevails and autonomous calcium activity is lower than in the cortex or hippocampus. Our data therefore reflect a specific role of SNr astrocytes in sensing the STN-GPe-SNr loops activity and suggest that SNr astrocytes could potentially feedback on SNr neuronal activity. These findings have major implications given the position of SNr in the basal ganglia network.

Carbon nanotube substrates are promising candidates for biological applications and devices. Interfacing of these carbon nanotubes with neurons can be controlled by chemical modifications. In this study, we investigated how chemical surface functionalization of multi-walled carbon nanotube arrays (MWNT-A) influences neuronal adhesion and network organization. Functionalization of MWNT-A dramatically modifies the length of neurite fascicles, cluster inter-connection success rate, and the percentage of neurites that escape from the clusters. We propose that chemical functionalization represents a method of choice for developing applications in which neuronal patterning on MWNT-A substrates is required.

Purpose: The aim of this study is to overcome tumour cell resistance that generally develops after administration of commonly used anti-cancer drugs, such as doxorubicin., Methods: Recently, cell penetrating peptides have been used for their ability to deliver non-permeant compounds into cells. One such cell penetrating peptide, maurocalcine, has been isolated from the venom of a Tunisian scorpion. Herein, we report the effects of doxorubicin covalently coupled to an analogue of maurocalcine on drug-sensitive or drug-resistant cell lines MCF7 and MDA-MB 231., Results: We demonstrated the in vitro anti-tumoral efficacy of the doxorubicin maurocalcine conjugate. On a doxorubicin-sensitive cancer cell line, the maurocalcine-conjugated form appears slightly less efficient than doxorubicin itself. On the contrary, on a doxorubicin-resistant cancer cell line, doxorubicin coupling allows to overcome the drug resistance. This strategy can be generalized to other cell penetrating peptides since Tat and penetratin show similar effects., Conclusion: We conclude that coupling anti-tumoral drugs to cell penetrating peptides represent a valuable strategy to overcome drug resistance.

Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.

The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent K(m) for exogenous ADP (250-350 mM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 mm) is independent of intrinsic MgATPase activity. However, the K(m) may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum.

The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA). It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (gamma) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes.