Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.     

Morphometric quantification of mitochondria in the two steroidogenic ovine luteal cell types

Progesterone secretion is regulated by different mechanisms in large and small steroidogenic ovine luteal cells. Large cells secrete approximately 7-fold more progesterone in an unstimulated state than small cells. Since cholesterol side-chain cleavage, which is catalyzed by an inner mitochondrial membrane enzyme complex, is a major rate-limiting step in progesterone synthesis, mitochondrial components were quantified in the two steroidogenic cell types throughout the estrous cycle. Corpora lutea collected on Days 4 (n = 4), 8 (n = 4), 12 (n = 5), and 16 (n = 6) of the estrous cycle were prepared for electron microscopy. Volume densities of cell types within corpora lutea and mitochondrial densities within cell types were estimated by point-counting; nuclear and cytoplasmic volume densities were estimated by planimetric analysis. A total of 570 micrographs (magnification 5300 X) were analyzed. Large cell volume density was unchanged during the cycle (35 +/- 1%) while small cell volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 20 +/- 3% on Day 12. Large cell mitochondrial volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 23 +/- 1% on Day 16 accompanied by an increase in cytoplasmic volume density such that nuclear to cytoplasmic ratio increased (p less than 0.05) from 1:14 to 1:34 between Days 4 and 16. Small cell mitochondrial volume density increased from 11 +/- 1% on Day 4 to 14 +/- 1% (p less than 0.05) for the rest of the cycle while the nuclear to cytoplasmic ratio remained at 1:14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r.

We have studied the biogenesis of the envelope of E. coli B/r by measuring the synthesis of protein in separated inner and outer membranes during the cell cycle. While total protein and bulk inner membrane protein were synthesized continuously and at an exponentially increasing rate throughout the cycle, bulk outer membrane protein was synthesized at a constant rate throughout the cycle with an abrupt doubling in rate occurring 10–15 min before division. A similar pattern was observed when the rate of synthesis of an individual protein, the 36.5K outer membrane protein, was measured directly in total cell lysates. Neither thymine starvation nor changes in gene dosage of exponential cultures affected the synthesis of outer membrane protein, indicating that the doubling in rate is not controlled by a gene duplication mechanism. Other findings, however, further indicate that outer membrane protein synthesis is regulated in some way. Thus the concentration of 36.5K porin per unit surface area remained constant as the surface area/volume ratio varied widely with growth rate. We also obtained direct evidence for an overall limitation on the rate of synthesis of bulk outer membrane proteins; when a new class of outer membrane proteins was induced, the rate of synthesis of other surface proteins was correspondingly reduced. On the basis of these results, we discuss a model in which the linear growth of outer membrane protein results from a limitation of outer membrane polypeptide synthesis at the translational level, reflecting the linear expansion of the underlying peptidoglycan layer in the envelope.     

Growth of the mitochondrial inner membrane in synchronous cultures of Tetrahymena pyriformis: an examination of phospholipid accumulation

Based on morphological evidence, mitochondrial inner membrane growth has been reported to be discontinuous in heat shock-synchronized Tetrahymena pyriformis. As a biochemical measure of membrane growth under these conditions, we have examined phospholipid accumulation in the cell. No marked modulation of the accumulation of any of the major phospholipids could be detected through the cell cycle. At least 89% of the cardiolipin in the cells is restricted to the mitochondria, and we have used it as a marker for the growth of the mitochondrial inner membrane. During the heat shock synchrony, cardiolipin accumulates uniformly in parallel with the exponential rate of increase of total cellular phospholipids. These results suggest that at least the phospholipid component of all membrane systems in the cell grow continuously and uniformly. Additionally, we have shown that the total phospholipid content of Tetrahymena increases by a factor of 2.4 per generation following a series of heat shocks. No such net overaccumulation is observed for protein content.     

Morphometric analysis of several intracellular events occurring during the vegetative life cycle of the unicellular algaPolytoma papillatum

Summary Quantitative electron microscopy of serial sections was used to study thePolytoma papillatum cell and some of its constituents (nucleus, chondriome, leucoplast) during its vegetative life cycle.The volumes of cells just entering into or passing through mitosis varied considerably and seemed to determine the number of subsequent division processes.Whereas a volumetric balance existed between the cell (100%) and the chondriome (8–9%) during the whole life cycle, there was a correlation between cell and nuclear volumes (8–10%) only during interphase growth and the onset of mitosis. At telophase the nucleus-to-cell-volume ratio was reduced to 2%, but gradually increased during cytokinesis (4.6% at early cytokinesis; 6.5% at late cytokinesis) until it reached the initial value again in newly formed daughter cells. The leucoplast-to-cell-volume ratio (10–26%) varied considerably without any recognizable dependence upon cell cycling.The mean short axis of mitochondrial profiles was proportional to the mean diameter (=thickness) of the mitochondria; the specific surface (outer membrane area per 100 m³ mitochondrial volume), and the surface-to-volume ratio changed rhythmically. Changes in mitochondrial surface-to-volume ratio (Sc/Vc) were apparently correlated with changes in mitochondrial diameter (Dc). This relationship can be approximately described by the function Sc/Vc=4/Dc.Deviations of the surface-to-volume ratios of the nuclei from the surface-to-volume ratios of idealized spheres of equal size, indicating profound changes in nuclear shape, were found mainly during mitosis.Results were compared with those obtained from other morphometric investigations and discussed with regard to their functional meaning.     

Changes in mitochondrial mass,membrane potential,and cellular adenosine triphosphate content during the cell cycle of human leukemic (HL-60) cells

Oxidative phosphorylation within the inner mitochondrial membrane generates the majority of cellular adenosine triphosphate (ATP) required for normal physiological functions (including regulation of cell volume and solute concentration, maintenance of cellular architecture, and synthesis of essential macromolecules). Its efficient functioning depends on the maintenance of an electrochemical gradient and is tightly coupled to the energetic demands of the cell and/or tissue. Commitment to and completion of the cell division cycle are sensitive to changes in the availability of mitochondrially derived ATP, although the relationship between cell cycle and mitochondrial physiology is poorly understood. Using vital, mitochondrial-specific fluorochromes to differentiate between mitochondrial mass (10-N-nonyl acridine orange) and mitochondrial membrane potential (Rhodamine123), together with a quantification of total cellular ATP levels, it was possible to generate profiles of these mitochondrial characteristics in HL-60 cells at different stages of their cell cycle. The data suggest that the availability of ATP changes in a cell cycle-specific manner and cannot be predicted by changes in mitochondrial mass or membrane potential. Furthermore, transition points in the cell cycle where ATP availability is low with respect to the amount of functional inner mitochondrial membrane have been observed. We suggest that these cell cycle phase transitions are sensitive to inhibition of mitochondrial activity because the basal levels of available ATP at these points are nearer to a theoretical “minimal threshold” below which cell cycle progression is inhibited. J. Cell. Physiol. 180:91–96, 1999. © 1999 Wiley-Liss, Inc.     

A role for mitochondrial aquaporins in cellular life-and-death decisions?

Mitochondria dominate the process of life-and-death decisions of the cell. Continuous generation of ATP is essential for cell sustenance, but, on the other hand, mitochondria play a central role in the orchestra of events that lead to apoptotic cell death. Changes of mitochondrial volume contribute to the modulation of physiological mitochondrial function, and several ion permeability pathways located in the inner mitochondrial membrane have been implicated in the mediation of physiological swelling-contraction reactions, such as the K+ cycle. However, the channels and transporters involved in these processes have not yet been identified. Osmotic swelling is also one of the fundamental characteristics exhibited by mitochondria in pathological situations, which activates downstream cascades, culminating in apoptosis. The permeability transition pore has long been postulated to be the primary mediator for water movement in mitochondrial swelling during cell death, but its molecular identity remains obscure. Inevitably, accumulating evidence shows that mitochondrial swelling induced by apoptotic stimuli can also occur independently of permeability transition pore activation. Recently, a novel mechanism for osmotic swelling of mitochondria has been described. Aquaporin-8 and -9 channels have been identified in the inner mitochondrial membrane of various tissues, including the kidney, liver, and brain, where they may mediate water transport associated with physiological volume changes, contribute to the transport of metabolic substrates, and/or participate in osmotic swelling induced by apoptotic stimuli. Hence, the recent discovery that aquaporins are expressed in mitochondria opens up new areas of investigation in health and disease.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Overexpression of mitochondrial uncoupling protein 2 inhibits inflammatory cytokines and activates cell survival factors after cerebral ischemia

Mitochondria play a critical role in cell survival and death after cerebral ischemia. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that disperse the mitochondrial proton gradient by translocating H(+) across the inner membrane in order to stabilize the inner mitochondrial membrane potential (ΔΨ(m)) and reduce the formation of reactive oxygen species. Previous studies have demonstrated that mice transgenically overexpressing UCP2 (UCP2 Tg) in the brain are protected from cerebral ischemia, traumatic brain injury and epileptic challenges. This study seeks to clarify the mechanisms responsible for neuroprotection after transient focal ischemia. Our hypothesis is that UCP2 is neuroprotective by suppressing innate inflammation and regulating cell cycle mediators. PCR gene arrays and protein arrays were used to determine mechanisms of damage and protection after transient focal ischemia. Our results showed that ischemia increased the expression of inflammatory genes and suppressed the expression of anti-apoptotic and cell cycle genes. Overexpression of UCP2 blunted the ischemia-induced increase in IL-6 and decrease in Bcl2. Further, UCP2 increased the expression of cell cycle genes and protein levels of phospho-AKT, PKC and MEK after ischemia. It is concluded that the neuroprotective effects of UCP2 against ischemic brain injury are associated with inhibition of pro-inflammatory cytokines and activation of cell survival factors.     

Identification of a novel protein MICS1 that is involved in maintenance of mitochondrial morphology and apoptotic release of cytochrome c

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.     

A high-order trans-membrane structural linkage is responsible for mitochondrial genome positioning and segregation by flagellar basal bodies in trypanosomes

In trypanosomes, the large mitochondrial genome within the kinetoplast is physically connected to the flagellar basal bodies and is segregated by them during cell growth. The structural linkage enabling these phenomena is unknown. We have developed novel extraction/fixation protocols to characterize the links involved in kinetoplast-flagellum attachment and segregation. We show that three specific components comprise a structure that we have termed the tripartite attachment complex (TAC). The TAC involves a set of filaments linking the basal bodies to a zone of differentiated outer and inner mitochondrial membranes and a further set of intramitochondrial filaments linking the inner face of the differentiated membrane zone to the kinetoplast. The TAC and flagellum-kinetoplast DNA connections are sustained throughout the cell cycle and are replicated and remodeled during the periodic kinetoplast DNA S phase. This understanding of the high-order trans-membrane linkage provides an explanation for the spatial position of the trypanosome mitochondrial genome and its mechanism of segregation. Moreover, the architecture of the TAC suggests that it may also function in providing a structural and vectorial role during replication of this catenated mass of mitochondrial DNA. We suggest that this complex may represent an extreme form of a more generally occurring mitochondrion/cytoskeleton interaction.     

The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase.

Bloodstream forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, bloodstream forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of bloodstream forms into the next stage of the life cycle of T. brucei.     

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.     

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Supramolecular organization of tricarboxylic acid cycle enzymes

We propose a spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon). The structure is based on an analysis of data on the interaction between tricarboxylic acid cycle enzymes and the mitochondrial inner membrane, as well as on data on enzyme-enzyme interactions. The alpha-ketoglutarate dehydrogenase complex, adsorbed along one of the 3-fold symmetry axes of the mitochondrial inner membrane, plays a key role in formation of the metabolon. In the interaction with the membrane, two association sites of the alpha-ketoglutarate dehydrogenase complex participate, placed on opposite sides of the complex. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleoside-diphosphate kinase. Succinate dehydrogenase, which is the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of the metabolon on the membrane. The molecular mass of the complex (without regard to succinate dehydrogenase) is 8 x 10(6) Da. The metabolon symmetry corresponds to the D3 point symmetry group.     

昆虫线粒体发生的生化和亚显微结构的研究

线粒体在细胞中的发生目前有各种观点的争论,其理论意义涉及到真核细胞的起源和进化、染色体和线粒体两个遗传体系之间的相互关系以及生物膜合成和组装机理等。我们对处于分化中的昆虫胸肌线粒体的观察结果是:(1)对粘虫变态期的呼吸和细胞色素氧化酶活力测定表明蛹期第8天的组织形成阶段是胸肌细胞分化和其线粒体发生的开始。电镜观察表明,线粒体形成分两个阶段:由颗粒结构(可能是酶蛋白与脂的复合体)装配成膜片和膜泡;由膜泡分化出内嵴,进而发育为线粒体。(2)QO₂值,P/O比和ATP酶活力的出现与膜结构的分化发育相平行。α-甘油磷酸氧化酶系统比谷氨酸氧化酶系统装配早;电子传递酶系比磷酸化酶系装配早。(3)蝗虫胸肌分化过程的电镜观察证明;先形成内膜小泡(直径约0.1微米左右),后形成外膜,组成简单线粒体;后者进一步分化发育为成熟线粒体。(4)QO₂值,P/O比和ATP酶活力与膜结构分化发育相平行。ATP酶的出现与能量转涣功能呈平行关系。膜形成早期和“幼稚”线粒体阶段,ATP酶尚未装配。(5)综合上述结果:线粒体膜由非膜结构逐步组装形成,线粒体内膜的各酶系组装次序不同步,线粒体DNA控制合成的膜蛋白在膜结构形成中似乎起核心和骨架作用;线粒体总组装过程在不同细胞中表现为多种途径和方式。     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.     

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.     

Anti-proliferative and pro-apoptotic effect of CPT13, a novel camptothecin analog, on human colon cancer HCT8 cell line

10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) is a novel semi-synthetic analogue of camptothecin, our previous report had shown that it possessed higher in vitro cytoxicity activity towards human colon cancer HCT8 cell line than topotecan. In this study, the anti-proliferative effect of CPT13 on HCT8 cell line in vitro was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of CPT13 towards HCT8 cell line, the cell cycle distribution, apoptosis proportion, the nuclei morphological changes and caspase-8 and caspase-3 activities were measured. Additionally the changes of mitochondrial morphology and membrane potential (DeltaPsim) were analyzed by atomic force microscopy (AFM) and flow cytometry, respectively. The results showed that CPT13 inhibited HCT8 cell growth by causing cell cycle arrest at G2/M transition and induced apoptosis, as evidenced by the typical apoptotic morphology such as condensation and fragmentation of nuclei and formation of apoptotic bodies. The changes of mitochondrial morphology, dose-dependently decrease in DeltaPsim and the enhancement of caspase-8 and caspase-3 activities were observed in different concentrations of drug treatment group. Our results suggest that CPT13 induces apoptosis by alternations of mitochondrial transmembrane depolarization, activation of caspase-8 and caspase-3. Therefore, CPT13 appears to be a potent drug against human colon cancer via induction of apoptosis and may be used as an alternative drug to therapy cancer.