ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA ISOLATED FROM KIDNEYS OF NORMAL AND LEAD-INTOXICATED RATS

Mitochondria isolated from kidneys of lead-intoxicated rats have been shown to have decreased oxidative and phosphorylative abilities. The purpose of this study was to determine whether these abnormal mitochondria would undergo ultrastructural transformation during controlled respiration in the absence of phosphate acceptor (State IV), as previously demonstrated for normal liver mitochondria. It was first shown that normal rat kidney mitochondria transforms from a condensed ultrastructural conformation to an orthodox conformation after 5 min of State IV respiration with pyruvate-malate substrate. Reversal to a condensed conformation follows stimulation of respiration with adenosine diphosphate (ADP). A large portion of kidney mitochondria from lead-poisoned rats do not change from condensed to orthodox conformation during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as these few orthodox mitochondria disintegrate. The conclusion is that those mitochondria that do not undergo change in ultrastructure have impairment of electron transport, and that those that do become orthodox have increased membrane lability and undergo degeneration.     

THE CONTROL OF PYRUVATE DEHYDROGENASE IN ISOLATED BRAIN MITOCHONDRIA

Abstract— The activity and control of the pyruvate dehydrogenase complex in isolated rat brain mitochondria has been studied. The activity of this complex in mitochondria as isolated from normal fed rats was 78 ± 10nmol.min⁻¹ mg mitochondrial protein⁻¹ (n = 18) which represented 70% of the total pyruvate dehydrogenase activity. The pyruvate dehydrogenase in isolated brain mitochondria could be inactivated by incubation in the presence of ATP, oligomycin and NaF. The rate of inactivation was dependent upon the added ATP concentration but inactivation below approx 30% of the total pyruvate dehydrogenase activity could not be achieved. The inactivation of pyruvate dehydrogenase in brain mitochondria was inhibited by pre-incubation with pyruvate. Reactivation of inactivated pyruvate dehydrogenase in rat brain mitochondria was incomplete in the incubation medium unless 10mM-Mg²⁺+ 1 mM-Ca²⁺ were added; NaF, however, prevented any reactivation (Fig. 4). It is concluded that the pyruvate dehydrogenase complex in rat brain mitochondria is controlled in a manner similar to that in other tissues, and that pyruvate protection of pyruvate dehydrogenase activity may be important in maintaining brain energy metabolism.     

OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.     

ROLE OF MALATE TRANSPORT IN REGULATING METABOLISM IN MITOCHONDRIA ISOLATED FROM RABBIT BRAIN

Abstract— Partly purified chromaffin granules were incubated in vitro with Ca²⁺ (with trace amounts of ⁴⁵Ca²⁺) in concentrations ranging from 4 μm to 1 mm. After incubation the granules were washed with media containing EDTA and then subjected to density gradient centrifugation (1.3 to 2.0 m-sucrose solutions) in order to characterize the particles which had taken up ⁴⁵Ca²⁺. By using marker enzymes and various inhibitors of Ca²⁺ uptake into such cell particles as mitochondria it was established that under the conditions of the experiments chromaffin granules took up Ca²⁺ from the incubation medium. To characterize this uptake a simplified density gradient procedure was tested and found to be suitable. The uptake of Ca²⁺ into chromaffin granules was strongly dependent on temperature. It was not activated by ATP. The uptake was linear up to 10 min. At high calcium concentrations (above 200 μm) the rate of uptake levelled off. The uptake at 37°C was 1 nmol Ca²⁺/mg protein/min at a Ca²⁺ concentration of 500 μm. Mg²⁺ had no influence on Ca²⁺ uptake, whereas Sr²⁺ (1 mm) inhibited it. The methods established in this study should prove useful for a further characterization of this Ca²⁺ uptake into chromaffin granules which is likely to represent a useful model for the Ca²⁺ uptake occurring in the intact gland.     

大豆磷脂脂质体对再灌注心肌线粒体的影响

利用Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流装置,研究在缺血－再灌注时补充大豆磷脂脂质体对心肌线粒体膜脂质特性和超微结构的影响。结果：在缺血－再灌注时补充大豆磷脂脂质体可提高线粒体膜磷脂含量,抑制胆固醇－磷脂摩尔比和膜脂质微粘度的增加,改善线粒体的超微结构。结果表明,补充大豆磷脂脂质体对再灌注心肌线粒体的脂质特性和超微结构的损伤性变化具有保护作用。     

Tree Species Effects on Calcium Cycling: The Role of Calcium Uptake in Deep Soils

Soil acidity and calcium (Ca) availability in the surface soil differ substantially beneath sugar maple (Acer saccharum) and eastern hemlock (Tsuga canadensis) trees in a mixed forest in northwestern Connecticut. We determined the effect of pumping of Ca from deep soil (rooting zone below 20-cm mineral soil) to explain the higher available Ca content in the surface soil beneath sugar maple. We measured the atmospheric input of Ca with bulk deposition collectors and estimated Ca weathering and Ca mineralization in the surface soil (rooting zone above 20-cm mineral soil) from strontium isotope measurements and observed changes in exchangeable Ca in soils during field incubation. Calcium leaching at 20 cm was calculated by combining modeled hydrology with measured Ca soil solution concentrations at 20-cm depth. We measured root length distribution with depth beneath both tree species. Calcium leaching from the surface soil was much higher beneath sugar maple than hemlock and was positively related with the amount of Ca available in the surface soil. Calcium leaching from the surface soil beneath sugar maple was higher than the combined Ca input from atmospheric deposition and soil weathering. Without Ca uptake in the deep soil, surface soils are being depleted in Ca, especially beneath sugar maple. More organically bound Ca was mineralized beneath sugar maple than beneath hemlock. A relatively small part of this Ca release was leached below the surface soil, suggesting that, beneath both tree species, most of the Ca cycling is occurring in the surface soil. Sugar maple had more fine roots in the deep soil than hemlock and a greater potential to absorb Ca in the deep soil. With a simple model, we showed that a relatively small amount of Ca uptake in the deep soil beneath sugar maple is able to sustain high amounts of available Ca in the surface soil. Received 20 June 2001; accepted 6 December 2001.     

MITOCHONDRIA IN LIVING CELLS: AN ANALYSIS OF MOVEMENTS

Time-lapse cinephotomicrography of mouse embryonic fibroblasts before and shortly after perfusion of tissue cultures reveals that the elongation of mitochondria caused by coenzyme A results from the terminal association of many shorter rods into a smaller number of long filaments. These are not permanent associations, but they reflect an exaggeration of the cohesive tendency of mitochondria, which in untreated cells is counterbalanced by frequent disjoinings and breakings of the anastomotic network. Our own observations and a survey of the literature suggest that elongate mitochondria with rapid movement and high metabolic activity tend to accompany proliferation in tissue cultures, and that mitotic inhibition of cultured cells may go together with short, slow mitochondria of low metabolic activity. The movement of mitochondria may be both active, reflecting metabolic exchanges with the cytoplasm, and passive, the result of hyaloplasmic currents.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE ACTH-INDUCED ULTRASTRUCTURAL TRANSFORMATION OF MITOCHONDRIA OF CORTICAL CELLS OF RAT ADRENALS IN TISSUE CULTURE

The ultrastructure of the mitochondria of the cultured cortical cells of rat adrenals was studied. In vivo it was found that the zona fasciculata mitochondria have vesicular internal structure. 600-A vesicles appear free in the matrix or as protrusions of the inner mitochondrial membrane. In tissue cultures of the fetal and newborn rat adrenal cortex it was seen that ACTH induces transformation of the tubulo-vesicular internal structure of the mitochondria to 600-A vesicles. Actinomycin D and puromycin inhibited this transformation if they were added with ACTH. When added alone, these inhibitors of protein synthesis induced no change in the ultrastructure of the mitochondria in cultured cortical cells of rat adrenals.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT-PURIFIED MITOCHONDRIA

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O₂ uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A-insensitive O₂ uptake in either gradient band. Antimycin A-insensitive O₂ consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O₂ uptake. This O₂ consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O₂ consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development.     

STUDY OF MITOCHONDRIA IN RAT LIVER : Quantitative Electron Microscopy

The electron microscope has been used to determine the weight distribution of isolated subcellular particles from normal rat liver. The following results are reported: (1) There exist at least two well defined weight populations of subcellular particles; their respective median weights are 1.3 x 10^-14 and 11 x 10^-14 gm. The lighter fraction is considered to consist of lysosomes, the heavier of mitochondria. (2) The mitochondrial fraction shows a log-normal distribution of the particle weight. (3) By the introduction of morphologic criteria, the mitochondrial fraction is divided into two groups, one consisting of a spherical, the other of an oblong type of particle. The data found support the following concepts: (a) Mitochondria increase their weight from a certain size up by linear growth. (b) Mitochondria divide. The division is not necessarily symmetric; in all cases, however, one part of the division product is a spherical particle. It is felt that these results constitute a valuable demonstration of the general capabilities of quantitative electron microscopy and may stimulate many other useful applications of this technique in cytology, bacteriology, and virology.     

植物钙素吸收和运转

近年来,钙素在植物体内的吸收和运输研究主要集中在细胞和分子水平,但整株水平上的研究也同样重要.整株水平上的钙吸收和运输包括根细胞的钙吸收、钙离子横向穿过根系并进入木质部、在木质部运输、从木质部移出并进入叶片或果实及在叶片或果实中运转分配等环节,既经过质外体也穿越共质体.钙离子通道、Ca2 -ATP酶和Ca2 /H 反向转运器等参与根细胞的钙吸收.在钙离子横向穿根进入木质部的过程中,需要穿越内皮层和木质部薄壁细胞组织.根系内皮层凯氏带阻挡了Ca2 沿质外体途径由内皮层外侧向内侧的移动,部分Ca2 由此通过离子通道流进内皮层细胞而转入共质体并到达木质部薄壁细胞组织,而由木质部薄壁细胞组织进入中柱质外体可能需要Ca2 -ATP酶驱动;还有一些Ca2 由内皮层细胞运出,沿内皮层内侧的质外体途径进入木质部导管,并通过导管运向枝干.钙离子以螯合态的形式在枝干导管运输;水流速率是影响钙离子沿导管运输的关键因子.钙离子在果实和叶片中的运输和分配不仅通过质外体途径也通过共质体途径.     

FORMATION OF MITOCHONDRIA IN NEUROSPORA CRASSA : A Quantitative Radioautographic Study

Cells of a choline-requiring mutant of Neurospora crassa, labeled with radioactive choline, were transferred to unlabeled medium. At various times during their subsequent logarithmic growth, a highly purified mitochondrial fraction was prepared by sucrose density gradient centrifugation, and the distribution of label among individual mitochondria was determined by quantitative autoradiography. Preliminary experiments indicated that, under the conditions of this "washout" experiment, choline served as a stable mitochondrial label. Radioautographic analysis showed that, in fully labeled mycelia and for three mass doubling cycles in the unlabeled medium, radioactivity was randomly distributed among all mitochondria; i.e., the distribution of autographic grains among individual mitochondria followed a Poisson distribution. In experiments in which pulse labeling for 10 minutes was used, the label was randomly distributed among all mitochondria. The data suggest that the mitochondrial mass is increased by a continuous process of addition of new lecithin units to the already existing mitochondrial framework.     

Energized Uptake of Sugars from the Apoplast of Leaves: A Study of Some Plants Possessing Different Minor Vein Anatomy

Solutions of sucrose, glucose, raffinose, and stachyose were fed via the petiole to detached leaves of plant species known to transfer sugars during photosynthesis into the phloem using either the apoplastic or the symplastic pathway of phloem loading. Symplastic phloem loaders, which translocate raffinose-type oligosaccharides and sucrose in the phloem, and apoplastic plants, translocating exclusively sucrose, were selected for this study. As the sugars arrived with the transpiration stream in the leaf blade within little more than a minute, dark respiration increased. Almost simultaneously, fluorescence of a potential-indicating dye, which had been infiltrated into the leaves, indicated membrane depolarization. Another fluorescent dye used to record the apoplastic pH revealed apoplastic alkalinization that occurred with a slight lag phase after respiration and membrane depolarization responses. Occasionally, alkalinization was preceded by transient apoplastic acidification. Whereas membrane depolarization and apoplastic acidification are interpreted as initial responses of the proton motive force across the plasma membrane to the advent of sugars in the leaf apoplast, the following apoplastic alkalinization showed that sugars were taken up from the apoplast into the symplast in cotransport with protons. This was true not only for glucose and sucrose, but also for raffinose and stachyose. Similar observations were made for sugar uptake not only in leaves of plants known to export sugars by symplastic phloem loading but also of plants using the apoplastic pathway. Increased respiration during sugar uptake revealed tight coupling between respiratory ATP production and ATP consumption by proton-translocating ATPase of the plasma membrane, which exports protons into the apoplast, thereby compensating for the proton loss in the apoplast when protons are transported together with sugars into the symplast. The extent of stimulation of respiration by sugars indicated that sugar uptake was not limited to phloem tissue. Ratios of the extra CO₂ released during sugar uptake to the amounts of sugars taken up were variable, but lowest values were lower than 0.2. When a ratio of 0.2 is taken as a basis to calculate rates of sugar uptake from observed maxima of sugar-dependent increases in respiration, rates of sugar uptake approached 350 nmol/(m² leaf surface s). Sugar uptake rates were half-saturated at sugar concentrations in the feeding solutions of about 10–25 mM indicating a low in vivo affinity of sugar uptake systems for sugars.     

ULTRASTRUCTURAL STUDIES OF ABSCISSION IN PHASEOLUS: LOCALIZATION OF PEROXIDASE

Segments of leaf abscission zone tissue of Phaseolus vulgaris L. cv. Red Kidney were fixed in glutaraldehyde, incubated to demonstrate peroxidase activity in medium containing 3,3'-diaminobenzidine (DAB) and postfixed in osmium tetroxide. Electron microscopic observation of treated tissue revealed pronounced deposition of highly electron-opaque material in the form of granules or globules in cell walls, on mitochondrial membranes, associated with rough endoplasmic reticulum and along the plasmalemma and tonoplast. This distribution pattern was typical of both non-treated and ethephon-treated tissue. Ethephon-treated material also contained these granules within cytoplasmic vacuoles. It is suggested that pH of the incubation medium may affect localization sites and that exposure of tissue to light during incubation may modify localization patterns. Differing patterns of distribution of the reaction product in treated and non-treated tissue may reflect changes in membrane permeability and microfibrillar modifications related to ethephon treatment.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: II. GRADIENT-PURIFIED MITOCHONDRIA LACK CYANIDE-INSENSITIVE RESPIRATION

Wheat leaves normally produced very little ethylene, but following a water deficit stress which caused a loss of 9% initial fresh weight, ethylene production increased more than 30-fold within 4 hours and declined rapidly thereafter. The changes in ethylene production were paralleled by an increase and subsequent decrease in 1-aminocyclopropanecarboxylic acid (ACC) content. The level of S-adenosylmethionine was unaffected, suggesting that the conversion of S-adenosylmethionine to ACC is a key reaction in the production of water stress-induced ethylene. This view was further supported by the observation that application of ACC to nonstressed leaf tissue caused a 70-fold increase in ethylene production, while aminoethoxyvinylglycine, a known inhibitor of the conversion of S-adenosylmethionine to ACC, inhibited ACC accumulation as well as the surge in ethylene production if the inhibitor was applied prior to the stress treatment. Cycloheximide, an inhibitor of protein synthesis, effectively blocked both ethylene production and ACC formation, suggesting that water stress induces de novo synthesis of ACC synthase, which is the rate-controlling enzyme in the pathway of ethylene biosynthesis.     

The Role of Calcium in Intracellular Cadmium Uptake by the Lichen Peltigera membranacea

The possible role of calcium in the intracellular uptake ofcadmium by the lichen Peltigera membranacea was investigated.Even when calcium was removed from extracellular sites by potassiumpretreatment, kinetic studies failed to provide evidence insupport of added calcium being a competitive inhibitor of cadmiumuptake. Calcium and cadmium uptake responded differently tothe presence of lanthanum, vanadate or verapamil. Except forthe effect of verapamil, intracellular of cadmium mostly reflectedthe quantity of cadmium bound to extracellular exchange sites.Copyright1993, 1999 Academic Press Peltigera membranacea (Ach.) Nyl., intracellular cadmium uptake, calcium uptake inhibitors, lichen     

The Uptake of Calcium and Strontium by Plants from Soils and Nutrient Solutions

Plants have been grown in soils and nutrient solutions containingdifferent Ca/Sr ratios to find out whether there is any biologicaldiscrimination between calcium and strontium. When tomato plants are grown in nutrient solutions containingmolar Ca/Sr ratios from 2/1 to 4,000/1, the shoots do not distinguishbetween the elements, but the roots absorb strontium preferentiallyat low concentrations of this element. Since ion exchange resins show the same phenomenon, though toa less marked extent, this is believed to be a physico-chemicalrather than a biological effect. No chemical reagent has been found which can extract calciumand strontium from soils in the same ratio as plants. Ammoniumacetate extraction may give misleading results for the plant-availableCa/Sr ratios in the soil. Data for the Ca/Sr ratios in barleyand lucerne grown on soils containing ratios from 3/1 to 650/1emphasize this point.