Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slightly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.This work was supported by grants of the Consiglio Nazionale delle Ricerche to both InstitutesThe present study is dedicated to Prof. Otto Bucher on occasion of his 65th birthday     

The induction of energized configurational changes in plant mitochondria,in vivo

Configurational changes in the mitochondrial membranes of the salt gland ofTamarix aphylla, which are dependent on the biochemical state of the mitochondria, are demonstrated. In the energized state the cristae expand and become closely associated. There is also an increase in the density of the matrix and a formation of strands of material in the matrix and between the closely associated cristae membranes. The energized condition can be discharged by incubation in a medium containing 2,4-dinitrophenol and the mitochondria are comparable to those observed in glands fixed by typical methods for electron microscopy.These studies were supported in part by Grant GB-8199 (W.W.T.) from the National Science Foundation.     

Preservation of the in vivo state of mitochondrial network for ex vivo physiological study of mitochondria

Over the course of many years our laboratory has been engaged in the study of physiological functions of mitochondria ex vivo.We showed that the unavoidable destruction of mitochondrial-reticular network during traditional isolation of the mitochondria diminishes the observable ex vivo changes of mitochondrial processes in vivo. Comparing preparations obtained from quiescent and stressed rats, we found that the great difference in size of assemblies of mitochondria preserved in homogenate disappears when it is diluted for the measurement of respiration. This also leads to a decrease in the difference between respiration of mitochondria from quiescent and stressed animals.We developed a new method that provides ex vivo stable preservation of the in vivo network using a cytochemical procedure on glass-adhered lymphocytes in blood smear. We radically changed the incubation medium for the measurement of dehydrogenase activity that excludes an artefact of succinate dehydrogenase hyperactivation ex vivo by non-physiological components of the traditionally used solution. Our method made it possible to observe ex vivo two- to eightfold increase in succinate dehydrogenase activity by adrenaline in vivo, while the activity of α-ketoglutarate dehydrogenase changed reciprocally.The data obtained show that the structure changes of the network play an important role in physiological regulation of mitochondrial functions. Thus, it may be possible to correct mitochondrial dysfunctions in the organism by substances supporting the stability of mitochondrial network. The developed method is non-invasive, informative and, therefore, is convenient for clinical investigations, particularly of mitochondrial diseases.     

Biochemical and haematological changes in HIV subjects receiving winniecure antiretroviral drug in Nigeria

Background

Hematological and biochemical abnormalities are among the most common clinicopathological manifestations of HIV patients on ART. Consequently, the development and assessment of indigenous antiretroviral drugs with minimal abnormalities becomes a necessity. The objective of this investigation was to assess potential haematological and biochemical abnormalities that may be associated with the administration of Winniecure ART in HIV patients undergoing treatment in Nigeria. Fifty (50) confirmed HIV positive ART naïve patients aged 36 ± 10 were observed for haematological and biochemical responses for 12 weeks. Haematological responses were assessed thrice at 6 weeks interval using coulter Ac-T differential analyser and biochemical indicators (bilirubin, creatine, urea, amylase, ALT, ALP, AST, albumin) assayed spectrophotometrically.

Results

The biochemical parameters ALP (P < 0.05), ALT (P < 0.0001), AST (P < 0.001) and amylase (P < 0.05) slightly increased at the 12^th week, no significant change was observed in plasma creatinine and urea concentrations while albumin levels decreased non-significantly (P > 0.002). Haematological results showed consistent reduction of ESR, eosinophil, absolute and differential lymphocytes, granulocytes and total WBC in the test subjects throughout the assessment period. Conversely, haemoglobin, platelet and PCV increased significantly (P < 0.05). At the 12^th week thrombocytopenia (10.30%) and anaemia (76%) were reduced to 2% and 31% respectively while neutropenia (4.2 to 8%), leucopenia (26.8 to 30%) and lymphopenia (1 to 10%) increased. No cases of neutrophilia, lymphocytosis, eosinophilia and leukocytosis was observed.

Conclusion

The drug has a reduced haematological abnormalities and normal kidney function was unaffected though there were signs of possible abnormal levels of hepatic enzymes beyond 12 weeks of treatment.     

Regulation of ion transport in mitochondria by respiratory chain enzymes and ATPase

The effects of the respiratory chain inhibitors as well as those of the inhibitors and substrates of ATP-synthetase in Ca2+ and K+ transport induced in the mitochondria upon the medium acidification in the presence of phosphate or arsenate, were investigated. Evidence has been obtained suggesting that under the experimental conditions used the transmembrane fluxes of K+ and Ca2+ are paralleled with H+ leakage through the proton channel of ATPase. It was found also that the system inducing cation fluxes at low pH values included peroxidation and hydrolysis of phospholipids. A scheme of regulation of ion transport in the mitochondria involving oxidative phosphorylation and oxidation and hydrolysis of lipids is proposed.     

阴道菌群改变与宫颈癌前病变相关性的初步研究

目的 本实验对130例健康、HPV感染[HPV（+）]及不同级别宫颈病变（CIN）患者阴道菌群进行研究分析,揭示阴道菌群变化与宫颈病变的发生及HPV感染的关系,为阻断宫颈病变的进一步发展提供理论依据。方法 收集2014—2015年大连市妇幼保健院门诊就诊的20~65岁患者130例,依据宫颈HPV检测和阴道镜下宫颈活组织检查病理诊断结果进行分组。采用QIAamp DNA mini和Blood mini试剂盒提取阴道菌群基因组DNA。利用PCR-DGGE技术对患者阴道菌群的变化进行分析,Quantity One软件进行聚类分析及凝胶测序法分析特异条带的种类。上述数据的处理和分析采用SPSS 19.0统计软件。结果 在DGGE图谱中,HPV（+）组阴道菌群丰度高于HPV（－）组。CIN组阴道菌群丰度较HPV（+）组升高。测序结果显示,HPV（－）组与HPV（+）组中3条共有强度较大的条带均属于厚壁菌门,HPV（+）组的特征谱带为阴道加德纳菌（Gardnerella vaginalis）。聚类分析和主成分分析（PCA）显示,HPV（+）组患者发生了菌群失调。结论 乳酸菌为阴道菌群中的优势菌;放线菌是HPV（+）的CIN患者的特殊细菌门,该菌属于阴道加德纳菌;HPV感染可引起阴道菌群稳态失调,且宫颈病变程度越重,阴道菌群失衡情况也相应加重。     

Effect of changes in the membrane lipid composition on the activity of respiratory chain enzymes in rat liver mitochondria

The lipid composition and fatty acid spectrum of individual phospholipid fractions of internal and external membranes of mitochondria was studied in alloxan diabetes. It was found that the phosphatidylserine content is reduced under these conditions, while those of lysophosphatidylcholines, diphosphatidylglycerols and cholesterol are increased, and the fatty acids are saturated with phospholipids. The observed changes in the lipid composition of membranes cause a decrease in the rate of oxygen consumption in various metabolic states as well as in the activity of NAD X H+-, succinate and cytochrome oxidases in rat liver mitochondria.     

Synchronization of the function of respiratory chain enzymes and ATP-synthetase in energized mitochondria

The present study revealed that the previously described effect of ATP-synthetase inhibition concomitant with inhibition of the respiratory chain functioning could be observed under different absolute values of delta phi on the mitochondrial membrane. This points out that the membrane potential is not a unique regulator in the coupling of the ATP-synthetase and respiratory chain activities. At the same time, we succeeded in obtaining some evidence testifying that under conditions of ATP-synthetase inhibition the amount of functioning respiratory chains has to be proportional the functioning of the ATP-synthetases units. The osmolarity of the incubation medium was shown to control the state of the oxidative phosphorylation system. The respiratory chain and ATP-synthetase should be considered as an enzymatic supercomplex only when the osmolarity is close to 150-300 mOsm (within the physiological range). The coupling effectivity (ADP/O) of mitochondria under these conditions is maximal. It is concluded that the respiratory chain and ATP-synthetase are tightly bound from the kinetic point of view. The ATP-synthetase inhibition induces proportional inhibition of the respiratory chain enzymes and vice versa, the respiratory chain inhibition induces proportional inhibition of ATP-synthetase.     

C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction

Micro-vesicles can be released by different cell types and operate as ‘safe containers’ mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway.In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.     

Stimulatory effects of calcium on respiration and NAD(P)H synthesis in intact rat heart mitochondria utilizing physiological substrates cannot explain respiratory control in vivo

Mitochondrial TCA cycle dehydrogenase enzymes have been shown to be stimulated by Ca(2+) under various substrate and ADP incubation conditions in an attempt to determine and understand the role of Ca(2+) in maintaining energy homeostasis in working hearts. In this study, we tested the hypothesis that, at physiological temperature and 1 mM extramitochondrial free magnesium, Ca(2+) can stimulate the overall mitochondrial NAD(P)H generation flux in rat heart mitochondria utilizing pyruvate and malate as substrates at both subsaturating and saturating concentrations. In both cases, we found that, in the physiological regime of mitochondrial oxygen consumption observed in the intact animal and in the physiological range of cytosolic Ca(2+) concentration averaged per beat, Ca(2+) had no observable stimulatory effect. A modest apparent stimulatory effect (22-27%) was observable at supraphysiological maximal ADP-stimulated respiration at 2.5 mM initial phosphate. The stimulatory effects observed over the physiological Ca(2+) range are not sufficient to make a significant contribution to the control of oxidative phosphorylation in the heart in vivo.     

Action of strong and weak nitrogenous bases on respiratory chain enzymes of mitochondria

The action of nitrogenous basis--electroneutral hydrazides (pK less than 7,50 and positive charged arylhydrazones (pK greater than 8)--on the respiratory chain enzymes and the influence of the electric charge and the size of alkoxylic group on biological activity compounds have been investigated. It has been shown that the size of alkoxylic group defines the selective action of nitrogenous basis on the enzymes of mitochondrial respiratory chain. The nitrogenous basis with a long alkoxylic group is shown to be inhibitors of NADH-dehydrogenase, their action is similar to rotenone. At the same time compounds with a short group are more effective in the inhibition of the enzymes of the initial segment in the respiratory chain mitochondria. The affinity of the organic cations of arylhydrazones to NADH-dehydrogenase is 100-1000 times higher than the affinity of electric neutral compounds.     

Malonaldehyde acts as a mitochondrial toxin: Inhibitory effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Malonaldehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines to modify proteins to inactivity enzymes and also modify nucleosides to cause mutagenicity. Mitochondrial dysfunction is a major contributor to aging and age-associated diseases. We hypothesize that accumulated MDA due to mitochondrial dysfunction during aging targets mitochondrial enzymes to cause further mitochondrial dysfunction and contribute to aging and age-associated diseases. We investigated the effects of MDA on mitochondrial respiration and enzymes (membrane complexes I, II, III and IV, and dehydrogenases, including alpha-ketoglutaric dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH)) in isolated rat liver mitochondria. MDA showed a dose-dependent inhibition on mitochondrial NADH-linked respiratory control ratio (RCR) and ADP/O ratio declined from the concentrations of 0.2 and 0.8 micromol/mg protein, respectively, and succinate-linked mitochondrial RCR and ADP/O ratio declined from 1.6 and 0.8 micromol/mg protein. MDA also showed dose-dependent inhibition on the activity of PDH, KGDH and MDH significantly from 0.1, 0.2 and 2 micromol/mg protein, respectively. Activity of the complexes I and II was depressed by MDA at 0.8 and 1.6 micromol/mg protein. However, MDA did not affect activity of complexes III and IV in the concentration range studied (0-6.4 micromol/mg protein). These results suggest that MDA can cause mitochondrial dysfunction by inhibiting mitochondrial respiration and enzyme activity, and the sensitivity of the enzymes examined to MDA is in the order of PDH>KGDH>complexes I and II>MDH>complexes III and IV.