Formation Of Giant Mitochondria Research Articles

The HMGB1 protein sensitizes colon carcinoma cells to cell death triggered by pro-apoptotic agents.

The HMGB1 protein has multiple functions in tumor biology and can act both as a transcription factor and as a cytokine. HMGB1 is released during cell death, and in our previous studies we demonstrated that HMGB1 induces a distinct, necrosis-like cell death in glioblastoma. In epithelial malignant tumors such as colorectal cancer (CRC), the HMGB1-dependent effects show cross-talk with apoptotic signal transduction. Treatment of CRC cells with low concentrations of recombinant HMGB1 results in dose-dependent cytotoxicity which is morphologically characterized by the formation of giant mitochondria and does not share features of apoptosis. HMGB1-triggered cell death is associated with intracellular ROS release, and overexpression of Bcl-2 blocks both the increase of ROS as well as HMGB1-dependent cell death. Importantly, treatment with recombinant HMGB1 or overexpression of endogenous HMGB1 strongly sensitizes CRC cells to the cytotoxic activity of the pro-apoptotic death ligand TRAIL as well as the small molecule Bcl-2 family inhibitor ABT‑737. Moreover, treatment of CRC cells with TRAIL or ABT‑737 induces a release of endogenous HMGB1 into the extracellular space, and preincubation with glycyrrhizin, an HMGB1 inhibitor, significantly inhibits induction of cell death by TRAIL and ABT‑737, suggesting that HMGB1 functionally contributes to the execution of cell death triggered by pro-apoptotic agents. Finally, we investigated the expression of HMGB1 in human CRC tumor samples and found that loss of HMGB1 expression is associated with a more aggressive phenotype and a more advanced stage of disease in patients with CRC. Altogether, our findings demonstrate a functional link between cytotoxic signaling cascades triggered by HMGB1 and pro-apoptotic agents leading to an HMGB1-dependent sensitization to CRC cell death. Thus, a further evaluation of recombinant HMGB1 as part of an experimental combination treatment of CRC seems warranted.

Abstract 2144: Characterization of HMGB1-induced cell death in colon carcinoma cells

Abstract The danger protein HMGB1 induces a distinct form of cell death in cancer cells which is different from classical necrosis, apoptosis, autophagy, or senescence. Interestingly, colon carcinoma cells are less sensitive for the cytotoxic activity of HMGB1 than other types of cancer cells. The aim of this study was to characterize the molecular and metabolic pathways regulating the susceptibility to HMGB1 in colorectal carcinoma. Stable ectopic over-expression of HMGB1 inhibited significantly the proliferation of colon carcinoma cells. Treatment of cells with recombinant human (rh) HMGB1 induced significant cytotoxicity in a concentration range of 80 – 200 nM. Cancer cells depleted of mitochondrial DNA were less susceptible to the cytotoxic effects of rhHMGB1. In ATP luciferase assays, after 72 h of incubation with rhHMGB1 a significant depletion of intracellular ATP was observed, paralleled by the formation of giant mitochondria. RhHMGB1 modulated the specific activity of the mitochondrial respiratory enzymes and the electron flux in the electron transport chain in a dose-dependent manner. Interestingly, addition of NAD+, L-pyruvate, or ATP did not protect the cells from rhHMGB1-induced cell death. RhHMGB1-induced mitochondrial and metabolic events were further characterized by tracking radioactive glucose and pyruvate isotopes. In summary, the HMGB1 protein induces a novel form of cell death in diverse types of cancer cells. In colon cancer cells, HMGB1 modulates the activity of the mitochondrial respiratory enzymes leading to a change in glycolytic flux and a strong depletion of intracellular ATP. These observations implicate a new functional link between the HMGB1 protein and mitochondrial respiratory regulation as well as glycolysis activity in cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2144. doi:10.1158/1538-7445.AM2011-2144

Danger Signaling Protein HMGB1 Induces a Distinct Form of Cell Death Accompanied by Formation of Giant Mitochondria

Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors.

Behavior of mitochondria, chloroplast and pyrenoid in synchronized cells of Chlamydomonas and Euglena

Summary: Giant mitochondria are temporarily formed, probably by fusion of smaller mitochondria, in Chlamydomonas reinhardi cells at an intermediate stage in the growth phase of the cell cycle. The formation of giant mitochondria is accompanied by a marked decrease in the oxygen-uptake activity of the cells and the division of giant mitochondria into smaller forms by a re-increase in the activity. Changes in the morphology of mitochondria of Euglena gracilis Z cells were also followed with an electron microscope during the cell cycle in a synchronous culture under photoautotrophic conditions. Giant mitochondria were temporarily formed, most probably by fusion of smaller forms, in the cells at an intermediate stage in the growth phase of the cell cycle. Furthermore, we found for the first time such phenomena in chloroplasts of Euglena gracilis Z, called?ggiant chloroplast?h, and we clarified their arrangement in the cell cycle. Changes in morphology of the pyrenoid and those in distribution of RuBisCO in chloroplasts were followed by immunoelectron microscopy during the growth and division phases of synchronized cells of Euglena.The immuno-reactive protein were densely localized in the pyrenoid, and thinly distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. From a comparison of photosynthetic CO2-fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO2-fixation in photosynthesis.

Effect of melatonin on the cardiotoxicity of doxorubicin.

This study was designed to investigate the preventive effect of melatonin on doxorubicin's most important side effect, cardiotoxicity. Forty male albino Wistar rats were utilized and the rats were divided into five groups: group I, 0.9% NaCl for 4 days; group II, doxorubicin 3 mg/kg/day for 4 days; group III, 2.5 % ethanol for 15 days; group IV, melatonin 6 mg/kg/day for 15 days; and group V, a doxorubicin and melatonin combination were administered intraperitoneally. At the end of the experiment, tissue samples obtained from the cardiac muscle of the left ventricle of the rats were processed for measurement of malondialdehyde and for electron microscopic examination. Malondialdehyde, a product of lipid peroxidation, was found to be significantly higher in the doxorubicin group. However, in the doxorubicin and melatonin combination group the level of malondialdehyde was decreased statistical significant. The histological examination revealed destruction of myofibrils, disorganization of sarcomeres, mitochondrial degeneration and formation of giant mitochondria and lipid accumulation in the doxorubicin group. Also, accumulation of filamentous structures in the sarcoplasma in some of the cells, structural changes in capillaries and an increase in collagen fibers forming bundles were observed. When melatonin was added to the doxorubicin treatment all structural changes were reduced. The cardiotoxic side effect of doxorubicin used as a chemotherapeutic agent and was probably developed as a result of suppression of the antioxidant system and lipid peroxidation. Therefore, it could be assumed that the addition of melatonin in the treatment of doxorubicin could prevent the cardiotoxicity of doxorubicin.

New insights into the mechanisms involved in renal proximal tubular damage induced in vitro by ochratoxin A

The mycotoxin ochratoxin A is a contaminant of human and animal food products. It is a potent nephrotoxin known to damage the proximal tubule. The aim of this work was to investigate the effects of ochratoxin A on a porcine renal proximal tubular epithelial cell line (LLC-PK1), and to identify sensitive endpoints revealing damage at the epithelial barrier level and at the molecular level. Cells exposed for 24 h to 5-10 microM ochratoxin indicated a clear damage to the intactness of the epithelial barrier, as shown by measurements of trans-epithelial resistance and zonula occludens-1 protein expression. At the mitochondrial level we observed alterations of the normal functions, such as an increase of the membrane potential, the formation of straight extensions, and the formation of giant mitochondria. At higher ochratoxin concentrations (50 microM), at which cytotoxicity assays revealed a significant toxicity, alterations of the cytoskeleton organization and induction of apoptosis were evident. In addition, we analyzed the expression of genes by using a cDNA macroarray. Our data indicate that ochratoxin-induced nephrotoxicity can be detected at the barrier and at the mitochondrial level at rather low concentrations, at which conventional cytotoxicity assays are unable to reveal toxic effects.

Giant mitochondria induced in rat pancreatic exocrine cells by ethanol and iron.

Mitochondria are sensitive indicators of cellular pathology. Under certain circumstances, these organelles respond to cellular insult by a marked increase in size, resulting in the formation of giant mitochondria (megamitochondria). Ethanol has been implicated in the induction of giant mitochondria in rat hepatocytes. Since ethanol is reported to affect pancreatic mitochondria, we examined this organ for evidence of mitochondrial giantism in rats administered ethanol and a relatively small amount of supplementary iron. Diets were administered via a chronically implanted gastrostomy catheter. Rats were segregated in four groups: 1) basic high-fat diet, 2) ethanol and a high fat diet, 3) carbonyl iron and a high-fat diet, and 4) ethanol and carbonyl iron combined with a high-fat diet. After the animals were on their respective diets for 16 weeks, specimens of pancreas were extirpated and processed for and examined by transmission electron microscopy. Mitochondria in rats on the basic high fat diet resemble those in untreated controls. With ethanol and a high-fat diet, some mitochondria in virtually every exocrine cell are profoundly altered. Such organelles, which are of normal size, have undergone rearrangement of their internal membranes, with three of four parallel cristae residing at one or both poles of spherical to ovate mitochondria. In rats receiving both ethanol and carbonyl iron, giant mitochondria are present in pancreatic exocrine cells. Except for their size, megamitochondria in many cases are virtual likenesses of the altered mitochondria in the alcohol-high-fat rats, having several stacked cristae at either pole and an enhanced matrix compartment. Many of the giant mitochondria have at least one expanded crista, which contains packets of helically coiled filaments. When control rats are fed carbonyl iron without ethanol, the pancreatic mitochondria display a marked propensity for forming clusters of tightly interlocked organelles, formations that may be a prelude to mitochondrial fusion. It appears that iron supplementation sets the stage for organelle fusion, hence enlargement, with the ethanol providing the stimulus for fusion to actually take place, and controlling the final morphology of the resultant megamitochondria.

Giant mitochondria in the mature egg cell ofPelargonium zonale

Giant mitochondrial nuclei (known as nucleoids or mt-nuclei), which contain extremely large amounts of DNA, were studied in thin sections of the mature egg and proembryo (2 and 6 days after double fertilization) ofPelargonium zonale. Samples were embedded in Technovit 7100 resin, stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by immuno-gold electron microscopic cytochemistry. The egg cell contained giant mitochondria (either long and stretched or cup-shaped) which contained a large amount of DNA (more than 4 megabase pairs). However, the other cells, such as synergids, the central cell and nucellus contained small spherical mitochondria. Giant mitochondria in the egg cell were often found to make mitochondria complexes due to the grouping of cupule-shaped mitochondria. Immuno-gold electron microscopic cytochemistry revealed that the mitochondrial DNA is localized in the electron transparent of the giant mitochondria. Apparently, the large mitochondria in the egg cell divided in stages to form small, spherical mitochondria during the early stages of embryogenesis and the DNA content in individual large mitochondrion also decreased significantly. The amount of mitochondrial DNA reached approximately 800 kbp in the globular embryo 6 days after double fertilization. The formation of giant mitochondria in mature eggs has significant aspects after double fertilization.

Host Ranges, Serology and Cytopathology of Eggplant and Tomato Strains of Eggplant Severe Mottle Virus, a New Potyvirus from Nigeria

AbstractTwo potyvirus isolates from the eggplant (Solanum melongena L.) cultivars ‘Ex Benin’ and ‘Ex Jos’, respectively, in Nigeria proved to be almost identical in host range, symptomatology and reactivity with antisera to various potyviruses. In eggplant they caused a severe systemic mottle, blistering and malformation of leaves and an abnormal serration of the leaf margins. A potyvirus isolate from tomato showing mosaic symptoms was similar, but not identical to the eggplant isolates. In the slide, precipitin test the serological differentiation indices were between 1 and 3 for the eggplant and tomato isolates. In the immunoelectron microscopical decoration test all three virus isolates showed some reactivity with antisera to the following potyvir, uses: dioscorea green banding mosaic, groundnut eyespot, a mungbean isolate of peanut stripe, pepper veinal mottle, telfairia mosaic and a tomato isolate from Taiwan. No reactions were observed with antisera to other potyviruses. Cytopathogenic effects w,ere similar for all three isolates in the arrangement of virus particles, the structure of the cylindrical inclusions and the occurrence of clusters of small vesicles. However, other cytological alterations like accumulations of rod‐shaped aggregates of,granular material, formation of giant mitochondria, degeneration of mitochondria and occurrence of a nucleolar inclusion differentiated the isolates.

Age and exercise-related changes in myocardial mitochondria in mice

A quantitative histochemical and an ultrastructural study was performed on the left ventricular myocardium of C57BL/6J female mice to evaluate the effects of aging and running exercise on mitochondrial structure and SDH activity. A comparison was made between 6-month-old ("young") and 27-month-old ("old") sedentary mice with age-matched mice subjected to running schedules (10 or 30 min/d) for 6 weeks. In longer-term studies, 27-month-old mice were given similar daily running schedules over a 10 month period ("long term runners"). No significant differences were found in SDH activity during the normal course of aging in mice (6 to 27 months of age), however, the response to running differed markedly in young versus old mice. "Young" trained mice showed significant increase in SDH activity compared with age-matched sedentary mice, whereas "old" trained mice showed significantly reduced SDH activity. Electron microscopy showed ultrastructural changes in mitochondria associated with aging including the development of large aggregations of mitochondria in subsarcolemmal and paranuclear sites. Running schedules, especially in aged runners, caused an increase in interfibrillar mitochondria hypertrophy, loss of matrix and cristae, incorporation of lipid inclusions and the formation of giant mitochondria. These abnormal mitochondrial changes are interpreted as being degenerative and possibly contributing to the reduced SDH levels found in cardiac myocytes of aged runners. Our results indicate that whereas regular exercise in young animals enhanced SDH activity, in aged mice it may be detrimental rather than beneficial.

The ultrastructural features of secretory cells of some endocrine glands in aging

In the electron microscopic investigation of the secretory cells of adenohypophysis, adrenal cortex, adrenal medulla and pancreatic islets from the adult and old Wistar male rats, certain age-related ultrastructural features have been found. Age changes appeared to be more pronounced in the thyrotrophs, somatotrophs and gonadotrophs of the adenohypophysis and in zona glomerulosa and zona reticularis spongiocytes of the adrenal cortex. They consisted of atrophy of the Golgi apparatus, appearance of the cytoplasmic vacuoles, lipid and lipofuscin granules, secondary lysosomes and damage of the inner mitochondrial membrane. Parallel to these, hypertrophy of the Golgi apparatus and rough endoplasmic reticulum, formation of giant mitochondria and presence of a great number of secretory cells in the cellular cytoplasm were noted in zona fasciculata spongiocytes and chromaffin cells of the adrenal medulla, and in beta cells of the pancreatic islets during aging thus evidencing for the adaptive changes in the ultrastructure of these cells. However, no appreciable age changes have been observed in the ultrastructure of the adrenocorticotropic cells of the adenohypophysis.

Further characterization of the growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells.

As an approach for a better understanding of the mode of action of rotenone on mammalian cells we have studied the proliferation properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in vitro in the presence of 2.5 microM rotenone and after removal of the inhibitor. Experiments on asynchronous cells showed a rapid cessation of cell division accompanied by increased glycolytic rate, reduced oxygen consumption, moderate increase in DNA content and a fair increase in protein and RNA content of the cultures. DNA histograms obtained by flow-cytometry revealed an accumulation of cells in the G2 and M phase of the cell cycle. Electron micrographs taken after a 24 h treatment of cells illustrated the formation of giant mitochondria and fragmented nuclei. In order to elucidate the dual effect of rotenone- inhibition of mitochondrial energy metabolism and of mitotic processes- the influence on cells of rotenone at different stages of the cell cycle was tested using Ehrlich ascites tumour cells enriched in G1, S and G2 by centrifugal elutriation. DNA histograms and [3H]thymidine labelling index curves of cells from the different fractions cultured in the presence of 2,5 microM rotenone indicated that in addition to the observed accumulation in G2 and mitotic arrest of cells, the cell cycle progression is delayed in G1 phase. This may be explained by an effect of the inhibitor on the respiratory chain. S phase cells seemed to continue the cycle for several hours at a rate comparable to that of controls. Recultivation experiments on rotenone-treated asynchronous cells in inhibitor-free medium confirmed that some cells reinitiate DNA synthesis without preceeding cell division. Thus it must be concluded that cells at all stages of the cycle are affected by rotenone, but the impairment of cellular metabolism becomes manifest and lethal as soon as the acute block at mitosis is abolished and cells reenter the cycle.

PATHOLOGY OF EXPERIMENTAL RADIATION PANCARDITIS

Twenty‐three rabbits were given a single dose of 3.000R X‐irradiation over the heart to study the correlation between the ultrastructural changes of myocardial mitochondria and succinic dehydrogenase activity. The study was made from 1 hour until 1 month after irradiation. The myocardial mitochondria showed, at 1 hour, regressive changes such as irregular arrangement, swelling, vacuolation, decrease and disappearance of cristae, formation of giant mitochondria, and whorled myelin figures. These changes became maximum at 12 hours‐2 days. Regeneration of mitochondria had begun at 2 days. At 4–7 days, structurally normal mitochondria had markedly proliferated accompanied by large mitochondria with concentrically arranged cristae and small mitochondria around the nuclei and beneath the sarcolemma of myocardial cells. The myocardial succinic dehydrogenase activity, on the other hand, showed the lowest value at 12 hours ‐ 2 days, and recovered almost to the normal value at 7 days. At 2 weeks ‐1 month when myocardial fibrosis developed, however, the value again mildly decreased. Cardiac death due to radiation therapy may be Initiated by the mitochondrial hypofunction in radiation myocardial fibrosis, because the Irradiation injures the mitochondrial membrane resulting in morphological and functional changes.

Ultrastructural changes of renal tubules in the riboflavin deficient mouse.

Ultrastructural changes of the tubular epithelium in the mouse kidney produced by dietary riboflavin deficiency were studied by electron microscopy and cytochemistry. In riboflavin deficient mouse kidney, the ultrastructural changes are localized to the pars recta of the proximal tubule. They comprise so called vacuolar degeneration on light microscopy, which consists of the formation of giant mitochondria and vacuoles. During the development of riboflavin deficiency, mitochondria decrease in number and enlarge in size through fusion. Sometimes they are larger than nuclei in size. The vacuoles observed in tubular epithelia are divided into two different groups according to their morphological characteristics and origins. One is derived from proliferated peroxisomes, and another from increased cytoplasmic bodies termed cytosomes and cytosegresomes. These increased vacuoles occupy almost all of cytoplasm. Cytochemical studies also reveal that these vacuoles are peroxisomes and lysosomes. These changes are reversible on supplementation with riboflavin.

Effects of diazepam on photosynthesis, respiration, rubidium uptake, and finestructure of Scenedesmus obliquus in synchronous cultures

Effects of diazepam (Valium) on photosynthesis, chlorophyll/photosynthesis ratios, respiration, uptake of rubidium ions, and ultrastructure of Scenedesmus obliquus synchronized by a light-dark regimen of 14:10 hrs were determined. 80 and 160 muM diazepam, added to the nutrient medium at the start of the light-dark change (i.e., start of the cell cycle) gradually reduced rates of photosynthesis, below the initial rates from the beginning of the experiment. Contents of chlorophyll, however, remained nearly unaffected. Consequently, the diazepam-treated cells had a higher chlorophyll/photosynthesis ratio--also with regard to respiration in order to calculate the gross photosynthesis. The occurrence of photorespiration cannot be assumed. The net influx of rubidium was slightly reduced by 100 muM diazepam 0.5 and 2.0 hrs after the start of the cell cycle and was strongly inhibited after 5 to 14 hrs. 80 and 160 muM diazepam caused separation of thylakoids, formation of giant mitochondria and enlargement of vacuoles.

Abnormal mitochondrial DNA molecules in megamitochondria from cuprizone-treated rats.

The formation of giant mitochondria was induced in the livers of rats maintained on a diet containing 1 or 2% cuprizone for two weeks. Mitochondrial DNA was extracted, purified from the livers and observed under the electron microscope. In the control rats, mitochondrial DNA was made of monomers of 5 μm and of catenated oligomers. On the contrary, in the treated rats mitochondrial DNA was made of molecules of 4.7 μm to 7 μm, which could be classified into subpopulations of 4.84 μm, 5.00 μm, 5.17 μm, 5.37 μm. These molecules seem thus differ from each other by deletion of one 0.17‐μm sequence or by addition of one or several copies of this sequence. Size analogy with the D loop is discussed. Catenated oligomers and about 5% unicircular dimers were observed in the treated rats.

Chondriosphere Formation and Their Degeneration in Alcoholic Myocardiopathy

Previously, we reported the formation of giant mitochondria or chondriospheres 1n the myocardium of rats fed alcohol diet and 15% ethanol ad lib. For the present report, twenty-four C57BL male mice were randomly divided into three groups: Group I was fed sweetened semisynthetic liquid alcohol diet in which 36 per cent of the calories were derived from alcohol; Group II was maintained on a similar diet but alcohol was isocalorically substituted by sucrose, and Group III was fed regular Guilford mouse chow diet and distilled water ad 11b for 25 weeks.

Etude spectrale � basse temperature chez Euglena gracilis Z en culture synchrone sur milieu lactate: Variation de teneur en cytochrome 556

Low temperature spectra are described for whole Euglena cells. Euglena growing in synchronous culture with lactate medium show a cyclic variation of cytochrome 556 content during each cellular generation. The greatest quantity of cytochrome 556 seems to coincide with the non-dividing phase of the cells, the phase in which the mitochondrial network is observed. On the other hand after treatment of the Euglena with antimycin A, a correlation exists between the formation of giant mitochondria and an increase in the quantity of cytochrome 556.

A New Cell-Type in the Impulse-Conducting Tissue of Rat Heart

The ultrastructural alterations induced in the contractile cardiac tissue of rats by chronic administration of alcohol have been reported previously. The present paper deals with the cardiotoxic effects of ethanol on the impulse-conducting tissue. Several groups of male simonsen rats were fed commercial Purina rat chow diet and 15% ethanol ad libitum for 55 weeks. The controls were maintained on chow diet and distilled water. For electron microscopy tissue samples from atrium were fixed in 2.5% Cacodylate-buffered Glutaraldehyde, post-fixed in 2% buffered Osmium tetroxide and embedded in Epon-Araldite.On EM examination, the myocardial cells of alcoholic rat showed profound ultrastrcutural changes involving the formation of giant mitochondria by a process of coalescence, coupled with the fragmentation of myofibrils and the dehiscence of the intercalated discs. The conduction tissue of the treated animals, however, revealed an unusual type of cell, which to my knowledge has not been reported before in the literature. This peculiar conduction cell was wedged in between the ordinary myocardial cells and was characterized by a few remnants of sarcomeres delimited by dark Z lines and the familiar thick and thin filaments. The glycogen granules were scanty and the few mitochondria were randomly scattered.