Effects of ethidium bromide,tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

Summary Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.     

Morphometry and ultrastructure of prismatic cristae in mitochondria of a crayfish muscle. A hypothesis of the structural principle

Approximately 40% of the mitochondria in the sphincter muscle of the crayfish vas deferens have prismatic-type cristae. In cross section, the angular cristae have either triangular or rhomboid profiles which are surrounded by a hexagonal array of electron-dense dots. In longitudinal section, these mitochondria exhibit both thick and thin parallel lines, which represent cristae and filaments, respectively. It is postulated that the matrix of the prismatic-type mitochondria is packed with rodlets composed of an electron-dense core and a less dense shell. Close packing of these rodlets results in the regular hexagonal dot array. Deletion of fascicles of 3 or 4 rodlets results in spaces with triangular or rectangular cross sections. Lining of these spaces with membranes results in cristae with triangular or rhomboid cross sections.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Succinate dehydrogenase localization in the mitochondria of the renal tubules of cold- and warm-blooded vertebrates

Using electron microscopic histochemical technique, studies have been made on the activity of succinic dehydrogenase in the kidneys of the cod Gadus morrhua and dog. It was shown that chelate granules indicating localization of the enzyme in the mitochondria of nephronal cells, concentrate mainly in two zones -- between the membranes and inside the cristae. This distribution of the enzyme implies the presence of two pools of succinic dehydrogenase in the mitochondria which are utilized at different stages of oxidative phosphorylation. Succinic dehydrogenase content of the cristae is lower in cod than in dog.     

Ethidium bromide inhibits mitochondrial phosphorylating oxidation.

Ethidium bromide, in addition to combination with mitochondrial nucleic acids, is a phosphorylation inhibitor during glutamate and succinate respiration by mitochondria. Exhaustive washing of ethidium bromide-treated mitochondria did not relieve the inhibition nor significantly decrease the amount of bound dye. Dialysis against a cation exchange resin at 3 degrees for 17 hr removed about 97% of bound dye. This restored phosphorylating capacity to that of untreated mitochondria which had also been dialyzed against the resin. Since state 3 respiration was diminished and state 4 was unaffected by the presence of the acridine dye, and since neither swelling of mitochondria nor release of latent ATPase was observed, then ethidium bromide was not an electron transport inhibitor nor an uncoupler of oxidative phosphorylation. Inhibition of metabolic processes by ethidium bromide may be due in part to depressed generation of mitochondrial ATP.     

四种淡水养殖鱼类血细胞的细微结构

四种淡水鱼的血细胞形态基本相似。红血球形态与其他低等脊椎动物基本相似。淋巴球绝大部分是小淋巴球:单核球数量较少;四种鱼的嗜中性白血球形态结构差不多,胞核多为蚕豆形,很少见分叶核,分叶一般也只有二叶,这与哺乳类显然不同;嗜酸性白血球的形态结构与其他脊椎动物基本相似;在少数血涂片中看到了嗜碱性白血球。       

Fine structure of the parotid gland in the crest-tailed marsupial-rat (Dasyuroides byrnei).

The parotid gland of Dasyuroides byrnei was examined by light microscopy, and transmission and scanning electron microscopy. The acini were composed predominantly of seromucous cells with a few mucous cells. The seromucous cells were light or dark cells containing acidophilic spherical granules of moderate to high electron density and had well-developed cytoplasmic organelles-ordinary mitochondria and large mitochondria with tubular cristae, RER with vesicular or tubular elements, and Golgi apparatus with lamellae, vesicles and vacuoles. The mucous cells had basophilic amorphous granules of low electron density, like those of ordinary mucous cells. The intercalated ducts were composed of simple cuboidal light cells having a few electron-dense granules. The striated ducts consisted of tall columnar light cells containing numerous vesicles and mitochondria with tubular cristae, the same as found in acinar seromucous cells.     

A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes.

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Ultrastructure of mitochondria apparatus of cardiomyocytes in apoptosis induced by long-term anoxia in rats

Apoptosis in cardiomyocytes was induced by incubation of pieces of cardiac tissues under condition of anoxia. Electronmicroscopic investigation detected previously unknown changes in mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In addition to a mitochondrial population characterized with irrigated cleared matrix, anoxia induced the appearance of an atypical and previously unknown population of small electron-dense cardiomyocyte mitochondria. They were characterized by unusual localization inside electron-light mitochondria ("mitochondria inside mitochondria"). The most part of mitochondria with the irrigated matrix are commonly characterized by unusual types of rearrangements of the inner mitochondrial membrane. Under anoxic conditions, the inner mitochondrial membrane formed electron-dense ordered structures. This is a spongy structure with cells of equal size. Results of our study are discussed in terms of conception of changes in mitochondrial reticulum ultrastructure during apoptosis.     

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate.

Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate. In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found. In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for 1 h in a Ca-containing balanced salt solution. Freezing of fresh whole bones in liquid N2 before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes. The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Stable alterations in HeLa cell mitochondria following ethidium bromide treatment

This study describes the isolation of three HeLa cell clones after exposure of HeLa cells to ethidium bromide (EB) in culture medium for either 14 days, or 14 days plusreexposure for 30 days. All three EB-induced clones differed from the parental HeLa cell in various physical properties of their mitochondria. The ratio of mitochondrial DNA component I to component II was altered in clone HeLa-2A. In addition, the cytochrome content of the respiratory chain a + a₃, b and C₁ decreased, while the cytochroms c content remained unchanged. The amount of cytochromes b and c₁; which were not reduced by KCN treatment, but were reduced by dithionite, increased in clone HeLa-2A. The ultrastructure of HeLa-2A cells revealed several alterations characteristic of EB treatment. Some mitochondria had enlarged profiles, a reduced number of cristae and a more lucent electron density of the matrix. Other mitochondria were tightly packed with cristae, which occasionally showed a whorled configuration. These changes were observed 4 months (20–25 passages) after the omission of EB from the medium.     

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate

Summary Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate.In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found.In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for l h in a Ca-containing balanced salt solution.Freezing of fresh whole bones in liquid N₂ before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes.The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Pulse fluorimetry study in polarized light of DNA-ethidium bromide complexes

In previous works, a quantitative analysis of the fluorescence anisotropy decay, based on a comparison of the experimental measurements with a Monte Carlo simulation of the excitation energy migration, has been shown to provide the value of the unwinding angle of the DNA helix, induced by an ethidium bromide (E.B.) molecule intercalation. In the present work some of the characteristics of the model used in the computation are reexamined: namely the influence of the direction of the E.B. electronic moment, and the influence of the dye distribution along the DNA helix are studied. The computations are compared with experimental results obtained with new experiments performed with calf thymus and micrococcus lysodeikticus DNA-E.B. complexes. It is found that the difference in base composition of these DNA does not influence the fluorescence properties of their E.B. complexes. Our study confirms the validity of the dye distribution obtained with the single adjacent excluded site principle. Reasonable values of the unwinding angle are obtained by assuming that the transition moment direction lies along the great axis of the E.B. molecule. The value of this unwinding angle is compared with other values proposed in the literature.     

Ultrastructural Changes during Swelling and Contraction of Mitochondria from Cold-hardened and Non-hardened Winter Wheat

Mitochondria isolated from both 2 and 24 C grown winter wheat (Triticum aestivum L.) undergo spontaneous swelling in isomolar KCI solutions, but only 24 C mitochondria exhibit a substrate-induced contraction response. Electron microscopic examination revealed that 24 C mitochondria have more clearly defined cristae, less matrix material, and are generally more electron-dense than 2 C mitochondria. During swelling, the matrix material of both 2 and 24 C mitochondria expands and the mitochondria become less electron-dense. After partial swelling, 24 C mitochondria contract upon addition of succinate, and regain structural characteristics similar to those of untreated mitochondria. In contrast, mitochondria from 2 C seedlings continue to swell after addition of substrate, and many of the mitochondria become irregular in shape and lose much of their matrix material. A comparison of results obtained from absorbancy measurements, electron microscopy, and a Coulter Counter indicate that swelling and contraction involve changes both in over-all volume, and internal structural characteristics of mitochondria from 2 and 24 C grown seedlings. Electron microscopic examination of shoot cells showed that mitochondria in 24 C grown seedlings possessed more recognizable cristae and greater internal organization than mitochondria in 2 C seedlings.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Dual Electron Microscopic Localization of Mitochondrial Creatine Kinase in Brain Mitochondria

Mitochondrial creatine kinase in brain mitochondria appears to be located at two different intramitochondrial sites. By using immunogold-labeling techniques, a peripheral immunoreactivity was localized between the two boundary membranes, while an additional, central immunoreactivity was found at the crista surface. The peripheral enzyme was accessible to the antibodies after treatment of the brain mitochondria with 100-300 μg digitonin/mg mitochondrial protein, which left 75% of the activity bound to the membranes. Electron microscopic analyses revealed that 43% of the labeled, peripheral creatine kinase was bound at those places where outer membrane vesicles remained attached to the inner envelope membrane, suggesting that the enzyme is in involved in contact formation between outer and inner mitochondrial membranes. Postembedding staining of mitochondria on thin sections of brain tissue or in the isolated state led to the observation of a second location of creatine kinase inside the mitochondria, along the cristae, which was not accessible to the antibodies in isolated, digitonin-treated mitochondria.     

线粒体可能参与鲫鱼精子发生中拟染色体的形成

运用电子显微镜观察了鲫鱼生精细胞发育过程中拟染色体的形成和解体,以及拟染色体和线粒体的关系。在精子细胞阶段之前的各期生精细胞中都存在拟染色体。仅在精原细胞中观察到拟染色体的形成过程。拟染色体的形成方式与其它鱼类中拟染色体的形成方式相似。在生精细胞的发育过程中,线粒体的形态和数量发生变化。在初级精原细胞阶段,线粒体较大,多为球形,嵴少,基质电子密度低。随着生精细胞的发育,线粒体逐渐变小,多为长条状,嵴多,基质的电子密度升高。拟染色体形成后往往与线粒体结合。与拟染色体结合的线粒体往往解体,部分或全部的外膜和内膜破裂以至消失。线粒体解体后,其中的物质可能会转移到拟染色体中[动物学报52(2):328-334,2006]。     

The ultrastructural localization of adenosinetriphosphatase and alkaline phosphatase activity in eosinophil leukocytes

Summary The previously undescribed localization of reaction products of adenosinetriphosphatase and of alkaline phosphatase in eosinophil leukocytes was demonstrated by cytochemical studies of the rat intestine. Alkaline phosphatase reaction product was found only in minimal amounts on the plasma membrane but was distinct on the nuclear membranes and outer compartment of mitochondria but not on the cristae. The Golgi membranes and the endoplasmic reticulum reacted but less intensely. The specific granules showed no alkaline phosphatase activity.The adenosinetriphosphatase reaction, on the other hand, was found on the plasma membrane, vesicular or tubular profiles of the endoplasmic reticulum and on the matrix of the specific granules. The crystalloid of the granules did not show any reaction.Recipient of a postdoctoral fellowship from the muscular distrophy association of Canada.     

Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release

Release of cytochrome c from the mitochondrial intermembrane space is critical to apoptosis induced by a variety of death stimuli. Bid is a BH3-only prodeath Bcl-2 family protein that can potently activate this efflux. In the current study, we investigated the mitochondrial localization of Bid and its interactions with mitochondrial phospholipids, focusing on their relationships with Bid-induced cytochrome c release. We found that Bid binding to the mitochondria required only three of its eight helical structures (alpha4-alpha6), but not the BH3 domain, and the binding could not be inhibited by the antideath molecule Bcl-x(L). Membrane fractionations indicated that tBid bound to mitochondrial outer membranes at both contact and noncontact sites. Bid could interact with specific cardiolipin species on intact mitochondria as identified by mass spectrometry. Like the binding to the mitochondria, this interaction could not be blocked by the mutation in the BH3 domain or by Bcl-x(L.) However, a cardiolipin-specific dye, 10-N-nonyl acridine orange, could preferentially suppress Bid binding to the mitochondrial contact site and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release. These findings thus suggest that interactions of Bid with mitochondrial cardiolipin at the contact site can contribute significantly to its functions.