Lipophilic Proteins of Mitochondria from Microaerobic and Aerobic Continuous Cultures of Saccharomyces cerevisiae

Products of the mitochondrial protein-synthesizing system have been labeled in vivo in the presence of cycloheximide in microaerobic cells and in cells from glucose-limited and glucose-repressed aerobic continuous cultures of Saccharomyces cerevisiae. Lipophilic proteins were extracted from labeled mitochondrial membranes with aqueous methanol and neutral and acidic chloroform-methanol solvents. In glucose-limited aerobic and microaerobic cells, about half of the total mitochondrial products were soluble in organic solvents; in contrast, almost all of the labeled products were extracted from glucose-repressed mitochondria. Only trace amounts of labeled product were formed in mitochondrial membranes of a petite mutant. Lipophilic proteins were examined by polyacrylamide gel electrophoresis under dissociating conditions. Most of the label was associated with components of apparent molecular weights 12,000, 14,000 and 16,000. The relative proportions of these species in mitochondrial membranes are dependent on the concentrations of oxygen and glucose in which the cells are grown.     

Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis. Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions

Summary The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains.The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants.Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants. At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected.Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K. lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane.     

LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH : II. Morphological Characterization of Intact Pseudobranch, Subcellular Fractions, and Plasma Membrane Substructure

The pseudobranch of the pinfish Lagodon rhomboides is an unusually homogeneous and structurally simple tissue, well suited to cell fractionation studies. Its principal cell type, closely related to the chloride cells of teleost gill, is characterized by numerous mitochondria in close association with abundant tubular invaginations of the plasma membrane. Other cytoplasmic organelles are rarely encountered. In broken fresh pseudobranch cells negatively stained with ammonium molybdate, a 40 Å particulate layer was observed on the intracellular surface of the tubular plasma membrane fragments. Nuclear (N), mitochondrial-light mitochondrial (M+L), and microsomal (P) fractions, obtained by differential centrifugation, were characterized by examination of fixed, embedded pellets and unfixed preparations negatively stained with ammonium molybdate and potassium phosphotung-state. Mitochondria, in orthodox configuration and retaining their outer membranes, were observed in M+L and N. Significant amounts of tubular, sheetlike, or vesicular membrane fragments were observed in all three fractions. Many such fragments, when negatively stained, showed the 40 Å particulate surface layer characteristic of plasma membrane invaginations, and in some cases 20-Å projections could be resolved on the opposite (extracellular) surface. Since these morphological observations, together with previously presented biochemical data, suggest a plasma membrane localization of Na⁺, K⁺-ATPase, the possible association of the enzyme with membrane projections is discussed.     

原子力显微镜检测过表达α-突触核蛋白引起的线粒体结构变化

目的：利用原子力显微镜等方法,明确α-突触核蛋白（α-synuclein, α-syn）过表达对线粒体的影响。方法：将腺相关病毒（AAV）载体介导表达的α-syn (AAV /α-syn)病毒包装颗粒,给大鼠脑内黑质区定位注射,建立α-syn过表达的大鼠模型,并以AAV介导表达的报告基因LacZ (AAV/LacZ)为动物对照组。提取大鼠黑质脑组织的线粒体,并通过JC1染色检测线粒体膜电势（ΔΨ）变化和Western Blot检测线粒体中α-syn蛋白量的变化;采用原子力显微镜观测线粒体表面结构的变化。结果：实验组大鼠黑质脑组织过表达α-syn基因,第16周时,Western blot显示线粒体中α-syn蛋白量增加约2倍;JC1染色发现ΔΨ下降;原子力显微镜观测可见线粒体体积增大,线粒体膜表面出现较多孔道样改变。结论：大鼠黑质脑组织过表达α-syn基因,第16周时,可导致α-syn蛋白在线粒体积聚;并可引起线粒体增大、膜形成孔道样改变、ΔΨ下降。     

Ultrastructure of Klebsiella O3 Lipopolysaccharide Isolated from Culture Supernatant: Comparison with Other Lipopolysaccharides

Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant, which was found to exhibit a very strong adjuvant activity in augmenting antibody response and delayed-type hypersensitivity to protein antigens in mice, was examined by electron microscopy. When negatively stained with uranyl acetate or ammonium molybdate, the KO3 LPS was found to consist principally of flat ribbon-like structures branching freely (average width 16 nm and average thickness 7 nm) and to contain a small proportion of spheres (diameter 20–50 nm), both structures covered with fine hairy structures (average length approximately 10 nm). When the polysaccharide of KO3 LPS was stained by the periodic acid-thiosemicarbazide-silver proteinate procedure, silver granules were deposited on the ribbon-like structures and around the spheres, suggesting that the polysaccharide moiety is located on their surface and that the fine hairy structures consist of the polysaccharide moiety. Comparison by means of preparations stained with uranyl acetate or ammonium molybdate showed that KO3 LPS isolated from the culture supernatant has structural features in common with KO3 LPS isolated from bacterial cells, Escherichia coli O9 LPS isolated from the culture supernatant, and E. coli O127 LPS isolated from bacterial cells. On the basis of the present results, schematic representations of the common physical structure of LPS were drawn; the fine hairy structures attach to the wide surface of the flat ribbon-like structures along their lateral margin.     

Mitochondrial Changes in β0-Thalassemia/Hb E Disease

The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.     

Mitochondrial inner membrane particles seen in sections ofin situ large amplitude swollen mitochondria in rhizodermal cells of cress (Lepidium sativum L.)

Addition of 5–40 mM sodium acetate to root explants of cress (Lepidium sativum L.) growing in a nutrient medium causes large amplitude swelling of mitochondria in rhizodermal cells. On the average, 10 mM sodium acetate causes a fourfold increase in mitochondrial volume, with 40 mM sodium acetate producing an up to tenfold increase in mitochondrial volume. During swelling, however, the mitochondrial membranes remain predominantly intact, and only the outer membrane occasionally appears to be broken. Two types of swelling can be observed: an overall swelling of mitochondrial matrices and a less frequent local swelling which leads to clearly different matrical regions. These regions may sometimes be separated by a septum formed from the inner mitochondrial membrane. After large amplitude swelling in 10 or 40 mM sodium acetate, the visibility of lollipop-like particles on the matrix side of inner mitochondrial membranes is strongly enhanced. These particles are suggested to be identical with mitochondrial inner membrane particles as visualized by negative staining. The distribution of these particles is described. Possible mechanisms which may cause enhanced visibility are discussed.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : I. THE SEPARATION AND IDENTIFICATION OF A MEMBRANE FRACTION FROM ISOLATED MITOCHONDRIA

Mitochondria isolated from rat liver and suspended in 0.44 M sucrose were disrupted by treatment with 0.3 per cent Na deoxycholate. The treated suspension was fractionated by differential centrifugation into a number of fractions and the respective pellets were examined in sections in the electron microscope. One of these fractions was found to consist of apparently membrane-bound (vesicular) elements. The difference between interfaces and membranes was discussed and the material of this fraction was found to meet stated requirements identifying it as membranous. A detailed study of the disruption process undergone by mitochondria in the presence of Na deoxycholate showed that the elements of this fraction were derived from structural elements assumed to be mitochondrial membranes. The findings thus demonstrate that mitochondria do possess membranes as defined and that these membranes can be isolated in a relatively pure form.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Enzymac characterization and lipid composition of rat liver subcellular membranes

1.

1. Mitochondria, inner and outer mitochondrial membranes, microsomes, smooth and rough endoplasmic reticulum membranes and plasma membranes were isolated from rat liver.     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f ). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data. Received: 16 July 1996 / Accepted: 2 December 1996     

Stereologic studies on mitochondrial configuration in different organs of the rat

Summary Mitochondria from different organs of the rat with configurations ultrastructurally resembling those of isolated mitochondria of known respiratory states have been subjected to Stereologic analysis. Mitochondria were examined from mossy fibers of the granular layer of the cerebellar cortex (condensed state), of the pericentral hepatocytes (orthodox state), and of heart muscle and parietal cells of the gastric fundus (transitional state). In order to study the relationship between mitochondrial compartments and the internal membrane a partition coefficient was introduced, which expresses the volume of the matrix (E_mm) and external compartment (E_ocm) respectively per unit surface area of internal mitochondrial membrane. The Stereologic parameters investigated, i.e. surface density of the mitochondrial membranes, volume density of the mitochondrial compartments and membranes, and partition coefficients generally agreed with the visual evaluation of mitochondrial ultrastructure. However, analysis of the coefficient of variation /x × 100% for E_ocm and E_mm has shown significantly greater variability in the mitochondria of the myocardium than in the gastric mitochondria, despite similar ultrastructure. It is suggested that Stereologic methods, like time-lapse cinematography, give a compound picture of configurational variation and of its plasticity.     

Nuclear-extranuclear interactions affecting oligomycin resistance in Aspergillus nidulans.

Summary The extranuclear mitochondrial oligomycin-resistant mutation ofAspergillus nidulans, (oliA1), was transferred asexually into four nuclear oligomycin-resistant strains of different phenotypes. In all four cases, the possession of the nuclear plus extranuclear mutation led to an increase in the in vivo level of oligomycin resistance. In two cases, the altered cytochrome spectrum and impaired growth ability determined by (oliA1) were suppressed by the nuclear mutations. In the third case, the in vitro oligomycin resistance of the double mutant ATPase was dramatically increased above that of either of the component single mutant strains, indicating a synergystic interaction between the nuclear and extranuclear gene products. In the fourth case, the double mutant became cold-sensitive.A new extranuclear mitochondrial oligomycin-resistant mutation (oliB332) is described. This mutant is phenotypically similar to, though not identical with, (oliA1) but is separable by recombination.A range of nuclear oligomycin-resistant mutants have been mapped. Despite presenting five distinctly different phenotypes, they all map at the same locus.     

Cytochemical studies of mitochondria. I. The separation and identification of a membrane fraction from isolated mitochondria

Mitochondria isolated from rat liver and suspended in 0.44 M sucrose were disrupted by treatment with 0.3 per cent Na deoxycholate. The treated suspension was fractionated by differential centrifugation into a number of fractions and the respective pellets were examined in sections in the electron microscope. One of these fractions was found to consist of apparently membrane-bound (vesicular) elements. The difference between interfaces and membranes was discussed and the material of this fraction was found to meet stated requirements identifying it as membranous. A detailed study of the disruption process undergone by mitochondria in the presence of Na deoxycholate showed that the elements of this fraction were derived from structural elements assumed to be mitochondrial membranes. The findings thus demonstrate that mitochondria do possess membranes as defined and that these membranes can be isolated in a relatively pure form.     

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Interpretation of intraspecific variability in mtDNAs of Aspergillus niger strains and rearrangement of their mtDNAs following mitochondrial transmissions

Physical and functional maps of mitochondrial DNAs of Aspergillus niger strains representing different mitochondrial DNA RFLP patterns were constructed and compared. In spite of the high similarity in the organisation of mitochondrial DNAs among examined strains, differences could be easily recognised by applying molecular markers, such as the different intron content of the cox1 genes, the sequence of the intergenic regions between the Met- and His-tRNA genes and downstream of the tRNA-Gly gene. Intraspecific mitochondrial transfers between the heterokaryon incompatible mitochondrial oligomycin-resistant A. niger strain, as the donor, and other A. niger-sensitive strains bearing different RFLP patterns resulted in oligomycin-resistant progeny possessing either rearranged or unchanged donor mitochondrial DNA and recipient nuclei. Since the intergenic marker sequences of mitochondrial DNAs turned out to be identical in the donor and the progeny, it can be assumed that the oligomycin-resistant progeny inherit the mitochondrial DNA of the donor strain; this may either remain unchanged or may be modified by a mobile intron of the cox1 gene of the recipient mitochondria.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Mitochondrial DNA in Candida pintolopesii, a yeast indigenous to the surface of the secreting epithelium of the murine stomach

Candida pintolopesii 108-1 is an indigenous yeast which colonizes the surface of the secreting gastric mucosa of mice. We have been exploring the aerobic respiratory capacities of this organism in reference to its capacity to colonize the stomach surface, an environment that could contain little oxygen for microbial growth. In this paper, we report mitochondrial DNA and membranes in cells of a strain of this yeast isolated from the gastric epithelium of a mouse and compare the findings with those made by other investigators in studies of Saccharomyces cerevisiae. Putative mitochondrial DNA was isolated from crude lysates of C. pintolopesii and S. cerevisiae as fluorescing bands in CsCl gradients containing 4',6-diamidino-2-phenylindole. The DNA from C. pintolopesii hybridized with a 32P-labeled DNA probe for the 21S rRNA gene encoded by mitochondrial DNA in S. cerevisiae. Postvital cell staining with 4',6-diamidino-2-phenylindole and rhodamine 123 revealed mitochondrial DNA and membranes, respectively, in the cytoplasm of intact C. pintolopesii cells. The staining patterns were generally similar to those reported for S. cerevisiae. Finally, structures similar to those reported to be mitochondria in electron micrographs of S. cerevisiae were seen in preparations of C. pintolopesii cells examined by transmission electron microscopy. These data confirm findings from studies of its respiratory capacity published earlier that a strain of C. pintolopesii isolated directly from its native habitat has functional mitochondria.     

Mitochondrial DNA in Candida pintolopesii, a yeast indigenous to the surface of the secreting epithelium of the murine stomach.

Candida pintolopesii 108-1 is an indigenous yeast which colonizes the surface of the secreting gastric mucosa of mice. We have been exploring the aerobic respiratory capacities of this organism in reference to its capacity to colonize the stomach surface, an environment that could contain little oxygen for microbial growth. In this paper, we report mitochondrial DNA and membranes in cells of a strain of this yeast isolated from the gastric epithelium of a mouse and compare the findings with those made by other investigators in studies of Saccharomyces cerevisiae. Putative mitochondrial DNA was isolated from crude lysates of C. pintolopesii and S. cerevisiae as fluorescing bands in CsCl gradients containing 4',6-diamidino-2-phenylindole. The DNA from C. pintolopesii hybridized with a 32P-labeled DNA probe for the 21S rRNA gene encoded by mitochondrial DNA in S. cerevisiae. Postvital cell staining with 4',6-diamidino-2-phenylindole and rhodamine 123 revealed mitochondrial DNA and membranes, respectively, in the cytoplasm of intact C. pintolopesii cells. The staining patterns were generally similar to those reported for S. cerevisiae. Finally, structures similar to those reported to be mitochondria in electron micrographs of S. cerevisiae were seen in preparations of C. pintolopesii cells examined by transmission electron microscopy. These data confirm findings from studies of its respiratory capacity published earlier that a strain of C. pintolopesii isolated directly from its native habitat has functional mitochondria.