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

Summary Chick embryo heart fragments in primary hanging-drop culture were treated with sodium fluoroacetate to induce inhibition of aconitate hydratase, a mitochondrial enzyme of the tricarboxylic acid cycle. The mitochondria were analyzed in the living myoblasts by phase-contrast time-lapse cinemicrography. The results were recorded in a 16 mm film. After 20–30 minutes contact of the cells with the inhibitor some mitochondria became thickened and swollen. The swelling was polymorphous, asynchronous and reversible; the same mitochondrion could swell and shrink many times. Some mitochondria seemed not to respond to fluoroacetate and remained rod-like. Mitochondria appeared the only cell components to be morphologically affected by fluoroacetate and the changes were specifically caused by the inhibitor. The type of mitochondrial swelling differed from the large-amplitude respiration-dependent swelling of the isolated mitochondria in vitro and from the configurational changes of isolated mitochondria associated with the respiratory states. The evidence pointed to a specific connection between the biochemical lesion caused by fluoroacetate and the configurational changes of the mitochondria. The mitochondrial swelling was to a large extent reversed by washing the cultures with Tyrode physiological saline solution and the reversal was further accentuated by incubation of the cultures in fresh nutrient medium.This work was supported by grants of the Consiglio Nazionale delle Richerce of Italy to both Institutes.     

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.     

Biochemical changes during the fermentation of Prosopis africana seeds for ogiri-okpei production

Biochemical changes during fermentation of seeds of Prosopis africana for production of ogiri-okpei, a food condiment popular among people of West Africa were studied. Fermentation resulted in a net increase in concentrations of total soluble sugars and free amino acids, both reaching a peak after 72 h of fermentation but declining thereafter. Corresponding increases were observed in amylase and protease activities, respectively. Lipase activity was observed to be very strong, increasing throughout the duration of fermentation. Analyses of amino and fatty acid composition using an amino acid analyzer and gas liquid chromatography, respectively, revealed a wide variety of amino acids including glutamine > cystine > arginine and the fatty acids stearic > Arachidic > linolenic > linoleic in the unfermented seed in the highest concentrations. Fluctuations in the concentrations of these compounds were observed during the fermentation. At the end of 96 h fermentation, glutamine > cystine > lysine and an unidentified fatty acid > arachidic > linolenic acids were found in the highest concentrations. Marked increases in composition with increasing period of fermentation were observed for Ca, P, K, Mn, and Z. Transformations of amino acids, fatty acids, and minerals during the fermentation of this seed revealed during this study will contribute towards the development of an industrial process for ogiri-okpei as well as an understanding of its contribution to the nutrition of its consumers.     

Catecholamine control of enzymes involved in isocitrate oxidation of rat liver mitochondria

Treatment of rats or liver homogenates with catecholamines (isoproterenol or noradrenaline) increased activities of both NAD+ -dependent isocitrate dehydrogenase and NAD(P)+-transhydrogenase (in the direction of hydrogen transfer NADPH----NAD+) with no change in NADP+ -dependent isocitrate dehydrogenase. These effects were realized via beta-adrenoceptors. Cyclic AMP mimicked the catecholamine action on incubation with liver homogenate. The effects of catecholamines and cyclic AMP were not additive.     

柠檬醛损伤黄曲霉线粒体生化机理的研究

应用生物化学方法并结合扫描电镜,研究柠檬醛掺入黄曲霉细胞,并通过损伤线粒体(Mt),导致抑制其生长的机理。结果表明,在药物致敏浓度时,菌丝体经该醛作用后,胞内Mt呈不规则增多,氧化还原反应系统受到破坏,与对照组相比,柠檬醛组的琥珀酸脱氢酶(Succinate Dehydrogenase,SDH)、苹果酸脱氢酶(Malate Dehydrogenase,MDH)活性分别呈不可逆下降271%和242%,随着药物浓度的升高,SDH、MDH的活性直至消失;以琥珀酸、α酮戊二酸和丙酮酸为底物时,线粒体呼吸速率分别下降24.1%、14.3%和36.1%,提示柠檬醛能使菌丝体DNA、RNA、脂类和蛋白质等生物合成受到抑制,促进细胞死亡。     

大血藤叶片生化成分的动态变化

对自然条件下大血藤叶片生化成分的动态变化进行了研究。结果表明;大血藤叶片可溶性糖,淀粉和总糖含量的动态变化有相同的趋势。在叶片生长季节早期含量较低。随着叶片的生长发育,其含量逐渐上升。至8月达到最大值后逐渐下降,叶片总糖含量与日均净光合速率的季节变化呈极显著的正相关,在整个生长季节中,可溶性糖含量比淀粉高,大血藤叶片总蛋白含量季节性变化呈单峰曲线,在展叶初期含量较低;随后迅速上升,至8月达到最大值后逐渐下降,叶片总糖含量与日均净光合速率的季节变化呈极显著的正相关。在整个生长季节中,可溶性糖含量比淀粉高。大血藤叶片总蛋白含量季节性变化呈单峰曲线,在展叶初期含量较低;随后迅速上升,在6月初达到最大值后逐渐下降,落叶前降至最低占,可溶性蛋白质含量的季节性变化曲线与总蛋白含量基本相似,DNA含量在叶生长初期大幅度上升,至6月达到高峰后迅速下降,以后基本趋向稳定,RNA在叶生长初期有所上升,峰值也出现在6月,以后缓慢下降,到9月降至最低值,在落叶前又有所回升,总核酸含量的季节变化与RNA变化相似,RNA/DNA比值在生长季节中出现3次高峰,大血藤叶片总黄酮含量季节性变化呈“双峰”型,第1高峰期在开花期的5月。第2高峰期在秋季的9月份。     

The effect of consuming different proportions of hummer fish on biochemical and histopathological changes of hyperglycemic rats

Hammour fish (grouper fish) are known to be of great nutritional value for human consumption, as their protein has a high biological value and contains all the essential amino acids. Grouper fish are also a good source of minerals, vitamins, and fats that contain essential fatty acids. Thus, the current study aims to know the effect of different proportion of hummer fish on biochemical and histopathological changes of hyperglycemic rats. Twenty-four (24) Sprague Dawley-strain male albino rats, which weighed 150 ± 10 g, were divided into four groups. One group served as the negative control (normal), while the others were rendered diabetic using alloxan. One of the diabetic groups was considered the positive control and fed a standard diet, whereas the remaining two groups were fed with a 20% and 25% hammour fish diet for 28 days. At the end of the experiment, blood samples were taken from all the rats, and their organs were removed and subjected to biochemical analysis. The results indicated that the group fed with the 25% hammour fish diet exhibited significantly lower levels of liver, kidney, and heart damage, along with lower levels of serum glucose, total cholesterol, triglycerides, LDL, GOT, GPT and ALP, as compared to the positive control. The urea and creatinine levels were significantly higher for the rats that were fed the 20% hammour fish diet than for those in the positive control. The histopathological study of the heart showed a slight improvement of the heart tissues with the increase of hammour fish intake compare to the positive control, while kidney of rat from group 4, which were fed 25% hammour fish, showed granularity of epithelial lining glomerular tufts.     

Antileishmanial activity and ultrastructural changes of sesquiterpene lactones isolated from Calea pinnatifida (Asteraceae)

Bioactivity-guided fractionation of antileishmanial active CH₂Cl₂ phase of MeOH extract from leaves of Calea pinnatifida led to isolation of two sesquiterpene lactones calein C (1) and calealactone C (2), which structures were stablished on the basis of spectroscopic analysis. Compounds 1 and 2 displayed potent activity against Leishmania amazonensis promastigotes with EC₅₀ of 1.7 and 4.6 µg mL⁻¹, respectively. Compound 2 presented low cytotoxicity for J774 macrophages and displayed activity against amastigote forms of L. amazonensis similar to miltefosine with CC₅₀ values of 31.73 and 27.18 µg mL⁻¹, respectively. Additionally, compounds 1 and 2 caused ultrastructural changes in promastigotes leading to a loss of their classical structural morphology, as evidenced by electron microscopy. Also compound 2 decreased the mitochondria membrane potential. To the best of our knowledge, this is the first occurrence of 1 and 2 in C. pinnatifida. The results obtained highlighted the importance of studying sesquiterpene lactones isolated from Calea pinnatifida in terms of antileishmanial activity, in order to understand the mechanism of action of the isolated compounds in promastigotes forms of L. amazonensis.     

2-Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl-CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.     

Phenotypic variation in respiratory metabolism and complementation of murine hepatic mitochondria

Summary Genetically standardized inbred mice exhibit interstrain differences in hepatic mitochondrial respiration rate, respiratory control, efficiency of oxidative phosphorylation with Krebs cycle substrates, and quantity of protein in the mitochondrial fraction. Mitochondria of females of strains with positive combining ability for postweaning growth mixed in vitro show complementation for respiratory traits. Mitochondria of males when mixed do not exhibit complementation. Mitochondria isolated from female mice respire more rapidly, are more tightly coupled and have higher phosphorylation efficiencies compared to those from male mice across all strains assayed. Direct evaluation of androgen effects on mitochondrial respiratory capacity supported this conclusion.Journal paper no. 4277 of the Arizona Agricultural Experiment Station. Supported in part by a University of Arizona Foundation fellowship (DRB)     

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.