OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA OF DEVELOPING RAT BRAIN

Abstract—

• 1 Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by oxygen electrode techniques in mitochondria prepared from developing rat brain.
• 2 Oxygen consumption, ADP/O ratios and RCR based on mitochondrial protein concentration increased with maturation. Of the substrates employed, succinate supported oxygen uptake best and malate poorest.
• 3 The addition of exogenous NAD to the mitochondrial preparation had no effect on rate of oxygen uptake.
• 4 Lack of change in ADP/O ratio in the presence of glucose, a tricarboxylic acid cycle intermediate (α-oxoglutarate), and ATP leads us to believe that there is no significant hexokinase activity in this preparation.

     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA FROM LIVERS SHOWING CLOUDY SWELLING

Using succinate and α-ketoglutarate as substrates, oxidative phosphorylation has been measured in mitochondria isolated from livers showing cloudy swelling. This cellular change was obtained by injecting rats with S. typhi murium toxin and guinea pigs with diphtheria toxin. It has been found that phosphorylation associated with the oxidation of either of these substrates was partially inhibited in mitochondria from livers showing cloudy swelling, while the oxygen consumption was unchanged. Thus, the P:O ratios for both succinate and α-ketoglutarate were lower in mitochondria from treated animals than they were in normal mitochondria. Dephosphorylation of ATP was not significantly modified in mitochondria from livers showing cloudy swelling as compared with normal controls. No dephosphorylation of AMP and G-6-P was observed either in normal mitochondria or in mitochondria from treated animals.     

THE INFLUENCE OF BILIRUBIN ON OXIDATIVE PHOSPHORYLATION AND RELATED REACTIONS IN BRAIN AND LIVER MITOCHONDRIA: EFFECTS OF PROTEIN-BINDING

1. The uncoupling of oxidative phosphorylation of liver mitochondria by bilirubin does not occur in the presence of equimolar quantities of human serum albumin. With brain mitochondria, however, albumin was not protective. 2. A similar protective effect of albumin for liver, but not for brain, mitochondria was observed in studies of the effects of bilirubin on the ³²Pi-ATP exchange reaction. 3. The latent ATPase of fresh brain mitochondria is activated by Mg²⁺ but only slightly by DNP. Bilirubin increased the Mg²⁺ stimulated ATPase activity in liver mitochondria but depressed this activity in brain mitochondria. These effects were uninfluenced by protein binding. 4. Isotope studies with [¹⁴C]bilirubin demonstrated that the affinity of brain mitochondria for albumin-bound bilirubin is not greater than that of liver mitochondria. 5. The greater toxicity of protein-bound bilirubin for brain mitochondria than for liver mitochondria might be related to the greater lipid content of brain mitochondria.     

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.     

CELL PROLIFERATION IN NEWLY TRANSPLANTED EHRLICH ASCITES TUMOR CELLS

• 1 Ehrlich ascites tumor cells collected from donor mice on the 5th day after inoculation were injected into the peritoneal cavity of new recipient mice.
• 2 Cell cycle times were drastically shortened by transplantation, for instance, the length of the cell cycle from 47 to 21.5 hr, and the duration of S from 26.5 to 16.5 hr.
• 3 Transplantation also caused a transient delay of cells in G₂ followed by a rapid acceleration and produced an immediate increase in the number of cells in DNA synthesis by about 5–8%.

     

SPHEROSOMES AND MITOCHONDRIA IN THE LIVING FUNGAL CELL

Study of living hyphae of Fusarium oxysporum Schlect., Fomes annosus (Fries) Cooke, Ceratocystis fagacearum (Bretz) Hunt, Basidiobolus ranarum Eidam, and Mycotypha microspora Fenner with phase contrast revealed that these fungi have spherosomes similar to those in vascular plants. The spherosomes are conspicuous in the hyphal tip, suggesting some function other than fat synthesis. It may be that the Woronin bodies reported by other workers are spherosomes. Mitochondria in these fungi are highly pleomorphic and exhibit saltatory movement. They often interact with nuclei in a manner suggesting close membrane contact.     

运动性疲劳状态下大鼠骨骼肌线粒体氧化磷酸化功能的研究

目的和方法 :以SD大鼠递增负荷力竭性跑台运动为运动性疲劳模型 ,分别测定运动后即刻骨路肌线粒体 :①呼吸链复合体Ⅱ Ⅲ电子传递与质子泵出比值 (H /2e) ;②以琥珀酸 (S)为底物的呼吸控制 :态 3呼吸速率(R3 )、态 4呼及速率 (R4 )、呼吸控制比 (RCR)和磷 /氧比 (P/O) ;②H ATPase合成活力 ,探讨疲劳性运动中线粒体氧化磷酸化功能改变的机理。结果 :力竭性运动后以S为底物的线粒体R4升高 2 1.10 % (P <0 .0 5 ) ;呼吸链复合体Ⅱ Ⅲ的总、净H 2e分别降低 8.5 3和 19.5 1% (均P <0 .0 5 )。底物的RCR和P/O呈显著降低 (均P <0 .0 5 ) ,而底物的R3则有所增加 (P >0 .0 5 ) ,H ATPase合成活力降低 16.68% (P <0 .0 5 )。结论 :线粒体质子漏增加 ,呼吸链电子传递与质子泵出偶联程度下降 ,氧化磷酸化脱偶联导致无效氧耗增多 ,可能是运动性疲劳状态下线粒体氧利用率下降的重要机制。     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

外源性线粒体DNA在细胞内的重建(简报)

近年来发现人类多种神经肌肉疾病存在线粒体电子传递链(electron transport chain,ETC)缺陷。由于线粒体在遗传上受核基因和线粒体基因双重控制,给确定ETC缺陷的来源造成困难。转线粒体DNA技术是线粒体同无线粒体DNA的细胞(ρ°cells)融合,形成转线粒体DNA细胞系(mtDNA-transferred cell line,也称cytoplasmic hybrids,简称cybrids),使病人的线粒体DNA(mito-     

微管与肿瘤细胞运动——免疫细胞化学研究

运用免疫细胞化学方法对三种不等恶性程度人结肠腺癌细胞株HT-29分化较好,呈上皮样,细胞排列紧密,呈聚集性生长;免疫细胞化学显示;微管不甚明显。SW480及SW620细胞呈梭形。细胞间松散,常呈单个生长,免疫细胞化学显示微管十分丰富,该种形态特征与原发肿瘤恶性程度一致,前为高分化结肠腺癌,后为高恶性度结肠腺癌,具较强转移潜能,加入TGFβ1后,三株细胞均呈生长加速,多见分裂相,但微管未见明显改变,说明细胞快速分裂并非肿瘤浸润的唯一原因,加入道诺红菌素作用后,SW480及SW620微管结构出现明显改变,表现为断裂,卷曲或呈颗粒状。提示道诺红菌素能破坏细胞微管成分,微管的破坏可能抑制肿瘤细胞的转移。     

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.     

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE,AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl₂ is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

OXIDATION AND PHOSPHORYLATION PROCESSES IN BRAIN MITOCHONDRIA OF RATS EXPOSED TO CARBON DISULPHIDE

Abstract— The effects of acute and long-term exposure to CS₂ on oxidation and phosphorylation processes in brain mitochondria of rats were studied. Although rats developed different symptoms of poisoning, depending on the type of exposure, the brain mitochondria of both groups of animals exhibited the same types of disturbances in oxidative phosphorylation. The main characteristic of these disturbances was the uncoupling of oxidative phosphorylation indicated by lower respiratory control indices due to stimulation of oxidation of respiratory substrates by mitochondria in the metabolic state 4. This effect was accompanied by a decreased P:O ratio and a lower ATP-Pi exchange rate. An inhibitory effect of CS₂ on the energy transfer processes is also suggested.
The observed changes in oxidative phosphorylation were more distinct in the case of acute poisoning, with a longer period of an uninterrupted exposure enabling a more complete tissue saturation with CS₂, than in the case of long-term exposure with shorter periods of intoxication within the day.     

ULTRASTRUCTURAL DISTRIBUTION OF CELL SURFACE ANTIGENS IN AVIAN TUMOR VIRUS-INFECTED CHICK EMBRYO FIBROBLASTS

The distribution of neoantigens in the surface membrane of avian tumor virus-infected chicken embryo fibroblasts was examined on carbon replicas of cell cultures using hemocyanin-labeled antibody. New determinants appearing on the cell surface of virally infected but not transformed cells are thought to be common with components of the viral envelope. These antigens were found to exist in a diffuse, random array on the dorsal cell surface, with a denser accumulation along the cell processes. In living cells, surface antigens are capable of several types of redistribution when activated by reaction with antibody. Leukosis virus-infected (non-transformed) cells showed two apparently independent modes of redistribution: a relocation of some antibody-related sites to the cell margin; or an involvement of essentially all sites in randomly dispersed aggregates. Viral antigenic sites on sarcoma virus-infected (transformed) cells, reacted with antibody, were able to produce weak marginal relocation; but revealed a more striking tendency to migrate to some central location. The centripetal coalescence thus formed resembles the "cap" noted in other systems. Prior aggregation into "patches" may not be a prerequisite for such cap formation. Tumor-specific surface antigen detection and mapping was attempted by this technique, but results were equivocal. An antigen possibly characteristic of rapidly dividing cells occurred in a sparse, diffuse fashion over the surface of morphologically distinct "round" cells.     

牵牛花受精前后卵细胞质体和线粒体及其细胞质DNA的变化

应用透射电镜研究了圆叶牵牛（Ｐｈａｒｂｉｔｉｓｐｕｒｐｕｒｅａ （Ｌ．） Ｖｏｙｇｈｔ）和大花牵牛（Ｐ． ｌｉｍ ｂａｔａ Ｌｉｎｄｌ．）卵细胞在受精前和受精后质体和线粒体的变化。观察到卵细胞在受精前高度液泡化,在核周及卵细胞周围的薄层细胞质中有质体和线粒体。质体含１～２ 个较大淀粉粒。线粒体多呈环形或杯状。卵细胞受精后,细胞质增多,质体的数量也明显增加。质体基质变得更加浓厚,并且普遍含有嗜锇的球体。合子中线粒体丰富,但缺乏卵细胞中那种环形或杯状线粒体,多呈圆形。细胞质ＤＮＡ检测的结果表明,卵细胞质ＤＮＡ荧光有大小和形状不同的两类,它们在细胞中随机分布。一类较大,呈环状,可能是线粒体中的ＤＮＡ显示的荧光;另一类小的点状荧光,可能大多是质体ＤＮＡ荧光,前者比后者多。卵细胞受精后,细胞质类核发生变化,表现在数量上明显地比受精前少。研究揭示了牵牛花合子中细胞质ＤＮＡ 减少的现象,说明了母系质体ＤＮＡ 在受精后可能被降解,提供了母系质体不传递到后代的可能机制。