THE MICROSTRUCTURE OF THE COMPOUND EYES OF INSECTS

The apposition eyes of two diurnal insects, Sarcophaga bullata (Diptera) and Anax junius (Odonata), have been examined with the electron microscope. In the latter case only the rhabdom is described. The rhabdom of the fly consists of a central matrix and seven rhabdomeres, one for each retinula cell. The rhabdomeres show an ordered internal structure built up of transverse tubes, hexagonal in cross-section. These slender compartments running the width of the rhabdomere are 370 A in diameter. After fixation with osmium tetroxide the walls of the compartments are more electron dense than the interiors. The retinula cells contain mitochondria, and pigment granules smaller than those found in the pigment cells. These granules tend to cluster close behind the membranes which separate the retinula cells from their rhabdomeres. The rhabdom of the dragonfly is a single structure which appears to be composed of three fused "rhabdomeres," each similar to a rhabdomere of Sarcophaga. Reasons are given for believing that the rhabdom may be the site of photoreception, as well as the organ for analyzing plane-polarized light, as suggested by other workers.     

ON THE RESPIRATORY QUOTIENT OF LUPINUS ALBUS AS A FUNCTION OF TEMPERATURE

The temperature characteristics for the oxygen consumption and CO₂-production of the germinating seeds of Lupinus albus were previously found to be different. It was predicted qualitatively that the respiratory quotient of the seed should be a function of temperature. A quantitative treatment is presented here, relating the change of the respiratory quotient with temperature and the temperature characteristics. Experimental results agree satisfactorily with the calculated value.     

OXIDATION-REDUCTION POTENTIALS AND THE POSSIBLE RESPIRATORY SIGNIFICANCE OF THE PIGMENT OF THE NUDIBRANCH CHROMODORIS ZEBRA

1. Oxidation-reduction potential methods have been applied to a study of the blue-purple pigment present in solution in the blood and in the tissue cells of the nudibranch Chromodoris zebra. 2. The blue-purple pigment and its yellow reduction product form a reversible system whose E_o'' = x0.102 volts at pH 7.0 and whose valence change from oxidant to reductant appears to be one. 3. The system is unlike oxyhemoglobin-hemoglobin in the mode of oxygen transfer. Its rôle as a possible respiratory material is discussed.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

ON THE MECHANISM OF DRUG HYDROXYLATION IN RAT LIVER MICROSOMES

The TPNH- and O₂-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed.     

A CYTOLOGICAL STUDY OF THE CENTRIFUGED WHOLE, HALF, AND QUARTER EGGS OF THE SEA URCHIN ARBACIA PUNCTULATA

While the ooplasmic components of centrifuged eggs of Arbacia punctulata do not stratify in homogeneous layers, we have obtained the following strata beginning with the centripetal end: lipid droplets, pronucleus, clear zone, mitochondria, yolk, and pigment. Whereas mitochondria may be found mingled with yolk bodies, we have never observed lipid droplets nor pigment bodies among any of the other inclusions. The so-called clear zone contains a heterogeneous population of inclusions: annulate lamellae, heavy bodies, Golgi complexes, and rod-containing vacuoles. The peripheral cortical granules of immature (germinal vesicle stage) and of mature eggs are not dislodged from the cortical ooplasm with the centrifugal force utilized. When the eggs are treated with urethane, prior to centrifugation, the cortical granules of mature eggs abandon their peripheral position. Further centrifugation of the initially stratified eggs produces nucleated and nonnucleated halves and the centrifugation of the halves results in quarters. The cytology of the halves and quarters is discussed. The halves and quarters have been activated with either sperm or hypertonic sea water. With the exception of the nucleated halves, we were unable to obtain plutei larvae from the other fractions (red halves and quarters). We believe that the lack of development of the various fragments is a function of the balance of particular inclusions necessary for differentiation.     

THE EFFECT OF IONIZING RADIATION UPON MITOCHONDRIA OF THE CENTRAL NERVOUS SYSTEM

After irradiation of rats with a linear electron accelerator, the respiratory rate in rat brain mitochondria was studied in the presence of substrate + ADP and after the conversion of ADP → ATP. After 20,000 rads of irradiation to the head there was a transient diminution of mitochondrial respiratory control when glutamate was used as the substrate, but no changes were observed when succinate was the substrate. Irradiation with 10,000 rads had no effect upon respiratory control. The addition of NADH₂ to irradiated mitochondria had no effect upon mitochondrial respiration. Irradiation of the brain with 20,000 rads failed to produce mitochondrial peroxidation or swelling, even in the presence of FeNH₄(SO₄)₂ or ascorbate. The slight changes in respiratory control of brain mitochondria following irradiation is in marked contrast to the susceptibility of mitochondria from other organs. The comparative radioresistance of brain mitochondria may be the result of greatly diminished radiation-induced peroxidation of cerebral mitochondrial membranes.     

THE PREPARATION AND PROPERTIES OF BACTERIAL CHROMATOPHORE FRACTIONS

Chromatophore material from the bacterium Rhodopseudomonas spheroides was freed of ribosomes by centrifugation in 27 per cent RbCl and then separated into "heavy" and "light" fractions by centrifugation through a sucrose gradient. The fractions differed from one another in the following ways. (a) The isopycnic density of the heavy fraction was between 1.15 and 1.18 gm/ml and that of the light fraction was 1.14 gm/ml. (b) The heavy fraction was able to bind ribosomes; the light fraction was not. (c) The light fraction was homogeneous in the ultracentrifuge and had a sedimentation constant, extrapolated to infinite dilution, of 153 s_20,w. The heavy fraction was grossly heterogeneous. (d) Both the amount of bacteriochlorophyll relative to protein and the ratio of bacteriochlorophyll to carotenoids were greater in the light fraction. (e) The spectra of the two fractions in the near infra-red were different. Comparisons of the chromatophore fractions from cells with different amounts of bacteriochlorophyll showed that the specific bacteriochlorophyll contents of the two fractions did not change to the same extent as did that of the whole cells. The amount of heavy fraction from pigmented cells was roughly independent of the cellular pigment content and was about equal to that from pigment-free cells. The amount of light fraction depended on the pigment content of the cells; no light fraction was obtained from cells devoid of bacteriochlorophyll. The cytochrome complements of both fractions underwent quantitative as well as qualitative changes with varying growth conditions. The size of the photosynthetic unit in R. spheroides appeared to increase as the total cellular bacteriochlorophyll content increased; however, the number of units per light fraction particle remained constant.     

THE EXTRACELLULAR RELEASE OF ECHINOCHROME

A study was made of the diffusion of the red pigment echinochrome from the eggs of the sea urchin, Arbacia punctulata, into sea water. Unfertilized eggs retained their pigment, over periods of hours. Outward diffusion of pigment from unfertilized eggs normally is entirely negligible, or does not occur at all. Enchancing the calcium or potassium content of the artificial sea water (while retaining isosmotic conditions) did not induce pigment release. Under anaerobic conditions, unfertilized eggs release pigment in small quantities. Fertilization alone brings about echinochrome release. Fertilized eggs invariably released pigment, whether in normal sea water, or sea water with increased calcium or potassium. This diffusion of the pigment began during the first cleavage, possibly soon after fertilization. The pigment release is not a consequence solely of the cell''s permeability to echinochrome (or chromoprotein, or other pigment combination) but is preceded by events leading to a release of echinochrome from the granules in which it is concentrated within the cell. These events may be initiated by activation or by anaerobiosis. The phenomenon was not due to cytolysis.     

Biochemical and ultrastructural properties of osmotically lysed rat-liver mitochondria

Isolated rat-liver mitochondria were osmotically lysed by suspension and washing 3 times in cold, distilled water. Pellets obtained by centrifugation at 105,000 g for 30 min were resuspended, fixed with glutaraldehyde and OsO₄, and embedded in Epon 812. Thin sections show the presence of two distinct membranous populations, each of which is relatively homogeneous in size and appearance. Swollen mitochondria (∼1.5 µ in diameter), which have been stripped of their outer membranes, are largely devoid of matrix and normal matrix granules and are referred to as "ghosts." The smaller (0.2 to 0.4 µ in diameter), empty appearing, vesicular elements, derived primarily from the outer mitochondrial membrane, can be differentiated from the ghosts on the basis of their smaller size and complete absence of internal structures, especially cristae. Each membranous element is enclosed by a single, continuous membrane; the "double membrane" organization typical of intact mitochondria is not observed. These findings indicate that the outer membrane of rat-liver mitochondria is spatially dissociated from the inner mitochondrial membrane by osmotic lysis of the mitochondria in distilled water. Three parameters of structural and functional significance in freshly isolated rat-liver mitochondria have been correlated with the structural alterations observed: (a) chemical composition (total protein, lipid phosphate and total phosphate), (b) specific and total activities of marker enzymes for mitochondrial matrix and membranes (malate dehydrogenase (MDH), D-β-hydroxybutyrate dehydrogenase (BDH) and cytochromes), and (c) integrated multienzyme functions (respiration, phosphorylation, and contraction). The data presented indicate that all mitochondrial membranes are completely conserved in the crude ghost preparation and that, in addition, about ⅓ of the matrix proteins (estimated by assays for MDH activity and protein) are retained. The study of integrated mitochondrial functions shows that a number of physiologically important multienzyme activities also are preserved in the water-washed preparation. The respiratory rate of ghosts per milligram of protein is 1.5 to 2.0 times that of intact mitochondria, which shows that the respiratory chain in the ghosts is functionally intact. The rate of phosphorylation is reduced, however, to about 25% of that measured in freshly isolated mitochondria and accounts for lowered P:O ratios using succinate as substrate (P:O ranges from 0.4 to 0.9). The phosphorylation of ADP to ATP is the only biochemical function, so far investigated, that is greatly affected by osmotic lysis. In addition, two lines of evidence suggest that the ghosts undergo an energy-dependent transformation resulting in contraction: (a) suspensions of the crude ghost preparation in 0.02 M Tris-0.125 M KCl medium show a marked increase in optical density upon the addition of ATP, and (b) ghost preparations incubated in ion-uptake medium in the absence of added calcium but in the presence of added ATP contain a large number of highly condensed ghosts (about 50% of the total profiles) when viewed as thin sections in the electron microscope. The correlated biochemical and morphological study presented here shows that the outer membrane of rat-liver mitochondria can be removed by controlled osmotic lysis without greatly impairing a number of integrated biochemical functions associated with the inner membrane.     

线粒体的热机效率原理及其在运动疲劳中的应用

基于呼吸链电子漏现象提出了用热机效率原理描述线粒体合成ＡＴＰ 的工作效率, 指出呼吸链漏电不仅使线粒体合成ＡＴＰ 的效率降低, 而且导致线粒体生成有害的活性氧自由基, 引起线粒体损伤。通过检测游泳耗竭小鼠心肌线粒体生成过氧化氢速率的增高和线粒体呼吸对氰化钾敏感性的下降,证明了耗竭运动引起呼吸链电子漏水平明显增高。随电子漏增加而出现的活性氧的损伤表现在线粒体脂质过氧化程度增加,呼吸链四个酶复合物的活性均有不同程度降低,以及呼吸控制率的下降等等。文章讨论了呼吸链电子漏和电子漏引起的活性氧生成对线粒体合成ＡＴＰ 效率的影响。     

MITOCHONDRIAL BIOGENESIS DURING DIFFERENTIATION OF ARTEMIA SALINA CYSTS

Mitochondria isolated from cysts of Artemia salina (brine shrimp) were found to be devoid of cristae and to possess a low respiratory capability. Hydration of the cysts induces marked biochemical and morphological changes in the mitochondria. Their biogenesis proceeds in two stages. The first stage is completed within 1 h and is characterized by a rapid increase in the respiratory capability of the mitochondria, their cytochrome oxidase, cytochrome b, cytochrome c and perhaps some morphological changes. In the second stage there is an increase in the protein-synthesizing capacity of the mitochondria as well as striking changes in mitochondrial morphology leading to the formation of cristae.     

ELECTRON MICROSCOPE HISTOCHEMICAL EVIDENCE FOR A PARTIAL OR TOTAL BLOCK OF THE TRICARBOXYLIC ACID CYCLE IN THE MITOCHONDRIA OF PRESYNAPTIC AXON TERMINALS

Respiration-linked, massive accumulation of Sr²⁺ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr²⁺ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO₂ into some of the pyruvate.     

ENZYME LOCALIZATION IN THE ANAEROBIC MITOCHONDRIA OF ASCARIS LUMBRICOIDES

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg⁺⁺-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

Gangliosides GM1 and GD1a normalize respiratory rates of rat brain mitochondria reduced by <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide

The rate of oxygen consumption by glutamate- and malate-energized rat brain mitochondria, which was stimulated by an uncoupler 2,4-dinitrophenol (DNP), declined in the presence of a prooxidant tert-butyl hydroperoxide. Preincubation with gangliosides GM1 or GD1a at micromolar (but not nanomolar) concentrations significantly slowed down this decline in the mitochondrial respiration, as shown by measuring absolute respiratory rates and ratios of the mitochondrial respiratory rate in the presence of DNP to the basal respiratory rate (V _DNP/V ₀). Gangliosides GM1 and GD1a also slowed down a decline in the DNP-stimulated mitochondrial respiration induced by long-term incubation (“aging”) of mitochondria on ice. The data obtained are likely to reflect a prooxidant-induced reduction in the activity of enzymes of the mitochondrial respiratory chain as well as a GM1- and GD1a-induced decrease in the degree of their inactivation. Interestingly, in the presence of the Trk receptor tyrosine kinase inhibitor (K252a) this effect of gangliosides was not manifested in any way. Our data suggest that the direct impact of gangliosides on mitochondrial signaling pathways, specifically on the Trk receptor tyrosine kinase, plays a certain role in the mechanism of their protective effect on cerebral neurons and, probably, neuroglia.     

THE FINE STRUCTURE OF PUROMYCIN-INDUCED CHANGES IN MOUSE ENTORHINAL CORTEX

Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the entorhinal cortex of puromycin-treated mice revealed the following: (a) Abnormalities were not observed in presynaptic terminals and synaptic clefts; many postsynaptic dendrites or somas contained swollen mitochondria. (b) Dispersion of polyribosomes into single units or condensation of ribosomes into irregular aggregates with loss of "distinctiveness" was noted in a few neurons 7–27 hr after puromycin treatment. (c) Cytoplasmic aggregates of granular or amorphous material were frequently noted within otherwise normal neuronal perikarya. (d) Mitochondria in many neuronal perikarya and dendrites were swollen. Mitochondria in axons, presynaptic terminals, and glial cells were unaltered. The relationships between these lesions and the effect of puromycin on protein synthesis and memory are examined. It is suggested that the disaggregation of polysomes is too limited to explain the effect of puromycin on memory. Special emphasis is given to the swelling of mitochondria. The possible mechanisms and the significance of this lesion are discussed.     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

Rat liver mitochondria allowed to accumulate maximal amounts of Ca⁺⁺ and HPO₄⁼ ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca⁺⁺ and HPO₄⁼ from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca⁺⁺-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca⁺⁺-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca⁺⁺-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca⁺⁺ and phosphate from the mitochondria into the medium.