Levels Of Respiratory Enzymes Research Articles

Metabolic profiling of myeloproliferative neoplasms reveals subtype specific metabolic traits

Introduction: Myeloproliferative neoplasms (MPN) are haematopoietic disorders leading to aberrant expansion of myeloid, erythroid, and megakaryocytic lineages, which are classified into three phenotypes namely Polycythaemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). The evolution of MPN mainly originates from three somatic mutations in haematopoietic stem cells (HSCs) including Janus Kinases2 (JAK2), Calreticulin (CALR), Myeloproliferative Leukemia Virus Oncogene (MPL). The JAK2 inhibitor Ruxolitinib, despite being the state-of-the- art therapy is unable to completely eradicate leukemic stem cells (LSCs). . Metabolic alterations are crucial in the development of cancer by creating different anaplerotic pathways supporting cancer cell proliferation. Metabolic vulnerabilities can be utilized to target MPN mutational hematopoietic stem cells. Methods: We preformed Seahorse analysis on three independent biological replicates for Jak2V617F wild type (WT), heterozygous (HET, represents ET) and homozygous (HOM, represent PV) c-kit positive cells and for even more primitive lineage negative, c-kit positive, Sca1 positive (LSK). We additionally used Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) and RNA sequencing to investigate metabolic differences in LSCs. Results: We see increased respiration in Jak2V617F HET c-kit cells compared to WT and Jak2V617F HOM cells. However, mitochondrial numbers as determined my mitotracker green flow cytometry are similar between all genotypes. Moreover, mitochondrial membrane potential, as measured my TRMT staining and flow cytometry, does not differ between genotypes in c-kit positive and in LSK cells. These data indicate that differences in respiration between genotypes is not due to changes in the mitochondrial compartment, but might instead be caused by changes in e.g. gene expression levels of respiratory enzymes or other players involved in cellular respiration. We therefore performed bulk RNA-sequencing on c-Kit positive and LSK cells. We found the glutamate receptor signalling pathway elevated in Jak2V617F Hom, with single cell RNASeq data from highly purified Hom Jak2V617F LSCs showing significant upregulation in glutathione metabolism, feeding into the same metabolic processes. To elucidate changes in metabolism in more detail, we generated data from two independent biological replicates of c-kit positive, murine cells from all three genotypes for LC-MS. We found that Jak2V617F Het cells show increased activity of the TCA cycle compared to Hom. This is in accordance with our Seahorse data showing increased respiration in Het compared to Hom. We therefore added an OxPhos inhibitor (IACS) to Het and Hom c-kit positive cells to characterise a possible vulnerability in the Het cells. Jak2V617F Het c-kit positive cells seem indeed more sensitive to IACS compared to Hom cells, where IACS yields little effect. In contrast, higher glutamate levels were discovered medium of Jak2V617F Hom cells after 24 hours of culture, indicating glutamate evacuation out of the cells. Conclusions Our data demonstrate, for the first time, differences in metabolic needs in subtypes of MPNs, with ET-like murine cells depending on the TCA cycle compared to PV-like murine cells, which deregulate the glutamate/ glutathione metabolism pathway.

Ecophysiological Influence on Scaling of Aerobic and Anaerobic Metabolism of Pelagic Gonatid Squids

We examined the oxygen consumption rates and activity levels of respiratory enzymes involved in the aerobic (citrate synthase [CS]) and anaerobic (octopine dehydrogenase [ODH]) metabolism of gonatid squids (Gonatus onyx and Gonatus pyrus) as a function of body size. The energy expenditure rates of gonatids (ranging from 2.51 to 8.79 micromol O(2) g(-1) h(-1) at 5 degrees C) are among the highest in Animalia when mass and temperature are taken into account. They reflect the low efficiency of jet propulsion and the animals' active life strategy as diel vertical migrants in the pelagic environment. Both metabolic rate and aerobic muscle potential (CS activity) were size independent across a size range of four orders of magnitude, which may be a result of their unusual body geometric allometry, extensive cutaneous respiration, and decreased energy-saving opportunities at larger sizes. Anaerobic metabolic potential (ODH activity) revealed a shift from positive scaling in juveniles to negative scaling among larger sizes. Juveniles are found in shallow water, where they are more susceptible to visually cued predators and require quicker size-specific escape responses and higher burst swimming capacities. Conversely, adults have reduced requirements for predator/prey interactions in the light-limited deep sea. Anaerobic metabolic scaling reflects an adaptive response to the changing physical and ecological demands across a depth gradient during this species's ontogenetic vertical migration.

Assessment of respiratory and ion transport potential of Penaeus japonicus gills in response to environmental pollution

The present study aims to pinpoint the respiratory and ion transport potential of gills of Penaeus japonicus living in Abu-Qir Bay, East of Alexandria, Egypt. Our results revealed clear histological impairments in gill structure. These alterations were mainly represented by the presence of large vacuoles in gill axis and gill lamellae. In addition, narrow, disrupted gill lamellae with wavy cuticle and shrunk pillar cells were detected. Moreover, some cells clearly showed pyknosis. Gill ultrastructure also showed abnormal chromatin condensation inside the nucleus. Obvious alterations in the typical shape and structure of mitochondria were observed. Noticeably, the main characteristics of ion regulating gill epithelium were absent thus suggesting a low ion transport activity of P. japonicus gills. Statistically, this was further proved by the significantly higher activity levels of respiratory enzymes, namely, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) compared to those of the ion transport enzymes, namely, adenosine triphosphatase (ATPase) and carbonic anhydrase (CA) in gills and haemolymph. SDH activity levels were higher than the corresponding levels of LDH in gills and its own level in haemolymph, indicating a contradictory effect of pollution on respiratory enzyme activity levels.

Short Hairpin RNA-mediated Silencing of PRC (PGC-1-related Coactivator) Results in a Severe Respiratory Chain Deficiency Associated with the Proliferation of Aberrant Mitochondria

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up-regulated in proliferating cells. Here, we investigated its in vivo role by stably silencing PRC expression with two different short hairpin RNAs (shRNA1 and shRNA4) that were lentivirally introduced into U2OS cells. shRNA1 transductants exhibited nearly complete knockdown of PRC protein, whereas shRNA4 transductants expressed PRC protein at approximately 15% of the control level. Complete PRC silencing by shRNA1 resulted in a severe inhibition of respiratory growth; reduced expression of respiratory protein subunits from complexes I, II, III, and IV; markedly lower complex I and IV respiratory enzyme levels; and diminished mitochondrial ATP production. Surprisingly, shRNA1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA4 transductants had normal respiratory subunit expression and a moderately diminished respiratory growth rate, both transductants showed markedly reduced growth on glucose accompanied by inhibition of G1/S cell cycle progression. Microarray analysis revealed striking overlaps in the genes affected by PRC silencing in the two transductants, and the functional identities of these overlapping genes were consistent with the observed mitochondrial and cell growth phenotypes. The consistency between phenotype and PRC expression levels in the two independent transductant lines argues that the defects result from PRC silencing and not from off target effects. These results support a role for PRC in the integration of pathways directing mitochondrial respiratory function and cell growth.

The Functional Organization and Control of Plant Respiration

The respiratory pathways of glycolysis, the tricarboxylic acid (TCA) cycle, and mitochondrial electron transport chain (miETC) are central features of carbon metabolism and bioenergetics in aerobic organisms. Respiration is essential for growth, maintenance, and carbon balance of all plant cells. Although the majority of respiratory enzymes are common to all organisms, plant respiration has evolved as a complex metabolic network endowed with a wide variety of unique characteristics. Plants have the option of employing alternative enzymes that bypass several of the conventional steps in cytosolic glycolysis, the TCA cycle, and miETC. The extent and conditions under which these bypasses operate is the subject of intensive research. The highly flexible nature of respiratory metabolism in plants has likely evolved in response to the crucial biosynthetic role played by respiration beyond its role in ATP generation; both functions must proceed if plants are to survive under varying and often stressful environmental and nutritional conditions. Additional complexity arises due to the existence of tissue- and/or developmental-specific isozymes of many plant respiratory enzymes, as well as the extensive interactions between photosynthesis and respiration, and plastidic, cytosolic, and mitochondrial metabolism in general. Recent progress in biochemistry, physiology, cell biology, genomics, transcriptomics, proteomics, metabolomics, and in vivo flux analyses have resulted in exciting new insights into many aspects of plant respiratory metabolism. Experiments on transgenic or mutant plants possessing significantly elevated or reduced levels of respiratory enzymes are augmenting our understanding of the functions, organization, and control of plant respiration. Metabolic engineering of plant respiration is of significant practical interest as it provides both an important approach to enhancing crop yields, as well as a potential mechanism for mitigating global climate change due to elevated atmospheric CO 2 levels. Referee: Dr. Greg C. Vanlerberghe, University of Toronto at Scarborough, Department of Life Sciences and Department of Botany, 1265 MilitaryTrail, Scarborough, ON Canada M1C 1A4

Determination of oxygen utilization pathways in an industrial strain of Saccharomyces cerevisiae during enological fermentation

It is generally admitted that, due to the low levels of respiratory enzymes and mitochondrial cytochromes, classical oxidative phosphorylation does not occur to any significant extent under alcoholic fermentation conditions. Under anaerobiosis, yeast growth normally requires oxygen in order to favour the synthesis of sterols and unsaturated fatty acids. However, in such conditions, superfluous oxygen consumption by yeast cells is observed. In this study, the potential relationship between this oxygen consumption and the known respiratory pathways of S. cerevisiae was investigated during enological fermentation. Partial and limited synthesis of mitochondrial cytochromes was observed during the growth phase. The superfluous oxygen consumed by the yeast cells appeared not to be related to classical respiration, but mainly to the operation of several mitochondrial alternative respiration pathways, which were linked to the cell cytochrome contents. It was further found that oxygen-dependent ergosterol biosynthesis accounted for less than 15% of the total oxygen consumption at the beginning of stationary phase in the absence of anaerobic growth factors. No significant oxygen consumption could be clearly attributed to fatty acids desaturation in the yeast cells.

Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments.

Upon exposure to oxygen, Escherichia coli increases the expression of enzymes essential for aerobic respiration, such as components of the TCA cycle and terminal oxidase complexes. This increase requires the elimination of repression mediated by the Arc regulatory system under anaerobic conditions. Coordinately, the synthesis of enzymes that function in anaerobic processes such as fermentation decreases, partly due to the inactivation of the transcription factor Fnr. E. coli is thus able to adjust the levels of respiratory enzymes to fit its environmental circumstances, and in this case, reduces the production of the less energy efficient fermentation enzymes in favor of the aerobic pathways. In contrast to the advantage in energy production, aerobiosis brings a disadvantage to E. coli: the production of reactive oxygen species (ROS), i.e. superoxide anion radical (O2.-), hydrogen peroxide (H2O2), and hydroxyl radical (.OH). These byproducts of aerobic respiration damage many biological molecules, including DNA, proteins, and lipids. To alleviate the toxicity of these compounds, E. coli induces the synthesis of protective enzymes, such as Mn-dependent superoxide dismutase (SodA) and catalase I (HP I), and this induction is controlled by the regulatory proteins SoxRS, OxyR, and ArcAB. Thus, ArcAB, Fnr, SoxRS, and OxyR function in concert so that E. coli can optimize its energy production and growth rate. Fnr and SoxRS are cytoplasmic, DNA-binding proteins, and these regulatory systems utilize iron-sulfur clusters as cofactors which may directly sense the redox environment. OxyR is also a cytoplasmic, DNA-binding protein, and appears to respond to redox potential through the oxidation state of a specific cysteine residue. In the ArcAB system (which belongs to the family of two-component regulatory systems), ArcB, a membrane protein, functions as the sensor, and ArcA, a DNA-binding protein, directly controls target gene expression. Under anaerobic conditions, ArcB undergoes autophosphorylation and transphosphorylates ArcA, stimulating ArcA's DNA-binding activity. During aerobic growth, the transphosphorylation of ArcA does not occur. In this signal transduction mechanism, the ArcB C-terminal or "receiver" domain plays a critical role; that is, it stimulates or abolishes the transphosphorylation depending on the metabolic state of the cell, which in turn is influenced by the availability of oxygen. E. coli thus employs at least four global regulatory systems which monitor the cellular oxidative/metabolic conditions, and adjust the expression of more than 70 operons to give the organism a better aerobic life.

The influence of training-detraining upon the heart, muscle and adipose tissue of female rats

The purpose of this study was to measure the effects of a 10 week training, 3 week detraining cycle upon heart, muscle and adipose tissue of the rat. Specific pathogen-free female Wistar rats, 175 g at the onset of the experiments, were separated into three treatment groups; Sedentary Control (SC), Trained (T) and Detrained (DT). Animals from the T group were killed at 2, 4, 6, 8, 10 weeks and animals from the DT group were killed at 7, 14 and 21 days after the last day of training. Unweighted swimming — 6 h/day, 5 day/week, was the form of training employed. The animals, after being sacrified, were anesthetized with nembutal (45 mg/kg body wt.) and muscle samples and heart removed. These tissues were frozen and analyzed at a later date for succinate dehydrogenase (SDH) activity (muscles), total protein (TP), total hydroxylprotein (TH) and wet and dryg weight (heart). Adipose tissue was removed last, digested in collagenase (5 mg/ ml) and the isolated cells used to measured 2-[ 3H]deoxyglucose uptake (DOG) and the conversion of D-[1- 14C]glucose (C-1) and D-[6- 14C]glucose (C-6) to CO 2. The results of this study show that 10 weeks of endurance training induced myocardial hypertrophy ( P < 0.05) which involved increases in both TP and TH, the heart of the trained animals having 20.8% more protein and a 28.5% more hydroxlprotein than the sedentary controls. With detraining hypertrophy was lost within 21 days. Training maintained fat cell size at its pre-trained diameter, while inactivity allowed growth in the adipocytes of the control animals. The uptake of DOG and the conversion of glucose C-1 and glucose C-6 to CO 2, were significantly ( P < 0.05) higher in the adipocytes of trained animals indicating that they were more responsive to insulin than the sedentary controls, which corresponded to increases in the respiratory enzyme levels of the muscles. During the first 7 days of detraining DOG uptake and both C-1 and C-6 glucose oxidation remained elevated. In conclusion the results of this study clearly demonstrate that there is a direct relationship between adiposity and training that can be related to the insulin responsiveness of the adipose tissue.

Effect of salt stress on the respiratory activity ofAspergillus sydowii

Respirometric studies with mitochondrial, fractions and whole cells revealed the presence of a more actively functioning respiratory system inAspergillus sydowii grown under salinity conditions. Oxidation of substrate, i.e., succinate, by the mitochondrial fraction was inhibited by the addition of rotenone, antimycin A, and cyanide. Electron microscopic observations ofAsp. sydowii grown in the presence of 2M NaCl indicated a comparatively larger size of mitochondria than in the control grown culture. A relatively larger fraction of the total cytoplasmic volume was occupied by the mitochondria in theAsp. sydowii grown in the media containing 2M NaCl. Levels of respiratory enzymes like succinate dehydrogenase. NADH dehydrogenase, cytochrome oxidase, NADH oxidase, and succinoxidase were higher in the culture grown in the presence of 2 M NaCl than in that grown in the absence of NaCl.

Relationship of Glucose Intolerance and Indocyanine Green Clearance to Respiratory Enzyme Levels in Human Cirrhotic Liver

The relationship of glucose intolerance and indocyanine green clearance to respiratory enzyme levels in liver mitochondria was studied along with standard liver function tests in 40 patients (8 cirrhosis, 19 cirrhosis with hepatoma, 13 non-cirrhotic with hepatoma). There was a negative correlation between cytochrome a(+a3) concentrations and phosphorylative activity per unit of cytochrome a(+a3) (r = -0.75, p less than 0.01), but no correlation between ICG-K and cytochrome a(+a3) concentrations. Cytochrome a(+a3) concentrations in cirrhotic patients with linear oral glucose tolerance pattern, characterized with no return toward normal glucose levels within 120 minutes after an oral glucose load, increased to 1.45 +/- 0.11 (10(-10) mol/mg of protein) compared with 0.90 +/- 0.07 in cirrhotic patients with parabolic OGTT pattern, characterized with a return toward normal glucose levels within 120 minutes (p less than 0.01) (0.82 +/- 0.02 in control patients without liver diseases). The former had high operative mortality regardless of ICG-K value and the latter had virtually uneventful clinical courses. It was suggested that increased cytochrome a(+a3) concentrations and impaired glucose tolerance might be responsible for decreased hepatic functional reserve and poor prognosis in cirrhotics.

Phenytoin, Electric, Ionic, and Metabolic Responses in Cortex and Spinal Cord

Post-tetanic potentiation (PTP) of monosynaptic reflex was estimated in spinal cords in the drug-free state after the administration of a convulsant dose of penicillin and after the administration of phenytoin. There was no apparent correlation between the degree of depression of PTP and the efficacy of controlling seizure activity by phenytoin. Extracellular potassium levels were measured with ion-selective microelectrodes. The post-stimulation clearing of [K+]0 was not accelerated by phenytoin, and frequently it was slowed. Post-stimulus undershooting of [K+]0 was diminished. Oxidation of NADH in cortex and of cytochrome a, a3 in spinal cord were measured by optical methods. Stimulus-evoked transient oxidation responses evoked by electrical stimulation were depressed by phenytoin. It is concluded that systemic administration of phenytoin in therapeutic doses does not stimulate Na+-K+-activated membrane ATPase in cortex and spinal cord. Unlike other depressants, phenytoin did not cause a reduction of "resting" redox levels of respiratory enzymes. The local regulation of blood flow remained unaltered after phenytoin administration. Phenytoin caused a moderate but consistent depression of the stimulus-evoked responses of potassium activity, electric potential, and oxidative enzymes, consistent with diminished outflow of potassium from cells, owing either to lesser activation of cells or to a lesser exchange of ions.

Effects of ethylenediaminetetraacetate and chloramphenicol on mitochondrial activity and morphogenesis in Mucor rouxii.

The present study demonstrates the importance of mitochondrial activities in controlling Mucor rouxii morphogenesis. The respiratory capacity of the spores of this facultatively anaerobic, dimorphic fungus becomes repressed if germination and growth take place in the absence of oxygen. The level of activity of mitochondrial enzymes such as cytochrome oxidase and malate dehydrogenase is lower in the anaerobic yeastlike cells than it is in ungerminated spores and in aerobic hyphae, but the reverse is true for glycolytic enzymes such as pyruvate kinase and alcohol dehydrogenase. Following exposure to air, yeastlike cells convert into hyphae after a lag period corresponding to aerobic adaptation. Anaerobic cultures grown in the presence of ethylenediaminetetraacetate (EDTA) at a concentration of 10(-4) M exhibit hyphal morphology. These cells, which are fully adapted to anaerobic fermentation, nevertheless have potentially active mitochondria with the same levels of respiratory enzymes as ungerminated spores. These cells are able to grow immediately after aeration, without an adaptation lag. Evidence is presented which indicates that the morphogenetic effect of EDTA is not the result of elimination of free metals. Additional evidence proving mitochondrial control of morphogenesis in M. rouxii is that chloramphenicol (4 mg/ml) induced the formation of respiratory-deficient, yeastlike cells in aerobic cultures.

Cardiac growth and respiratory enzyme levels in male rats subjected to a running program.

Cardiac growth and respiratory enzyme levels in male rats subjected to a running program.

Glucose-induced crypticity toward succinate metabolism in Saccharomyces lactis.

Saccharomyces lactis grown on glucose adapted very slowly to growth on succinate. This initial inability of glucose-grown cells to grow on succinate was paralleled by their inability to oxidize succinate. The possibility that repression by glucose of respiratory chain components was responsible for these observations was examined. Glucose-grown cells were able to respire glucose, ethyl alcohol, and lactate and were able to initiate growth on ethyl alcohol as rapidly as succinate-grown cells. Respiratory enzyme levels were essentially the same in cells grown on succinate or on glucose. Spectroscopic analysis revealed that glucose-grown cells possessed a full complement of cytochrome bands. Since by these criteria glucose-grown S. lactis appears to possess a competent respiratory system, the penetration of succinate-2,3-(14)C into succinate- and glucose-grown cells was examined directly. Glucose-grown cells exhibited a strong permeability barrier to succinate. Comparison of glucose oxidation by S. lactis and by S. cerevisiae suggests that the crypticity to succinate does not depend upon a strong Crabtree effect in S. lactis.

Changes in the osteoblastic and mitochondrial population of aging periosteum.

EARLY investigators1–4 interested in the physiology of bone have described filamentous, rod- and granular-shaped mitochondria in osteoblasts from a variety of animals both in vivo and in vitro. An apparent relation between the number of mitochondria and osteoblastic activity was also pointed out. Pritchard5 arrived at the same conclusion. Within the past decade numerous biochemists have revealed a good many of the substrates and enzymes involved with intermediary metabolism associated with mitochondria. More recently, cytochrome oxidase and succinic dehydrogenase activity of the periosteum was studied in normal and traumatized rat femora from birth to old age6,7. The results showed high levels of activity of respiration becoming more elevated at the period of maximum rate of bone formation. Following this period of intense activity (5 weeks of age), a sharp drop occurred to a low level, which was maintained throughout the life of the animal. Trauma to the femora stimulated a response of respiratory activity, however, the response becoming progressively less with increasing age. Changes in respiratory enzyme-levels are fundamental to the activity of cells in general, because these enzymes are part of the aerobic hydrogen transport system from which rich-energy-bond phosphate is generated.

Comparison of respiratory enzyme levels in tissues of mammals of different sizes.

SummaryHeart, kidney, liver, and brain of the cow, rat, and mouse were assayed for maximal activity of succinoxidase and malic dehydrogenase. The levels of the two enzymes generally reflect the inverse relationship of metabolism with body size in that the tissues of the cow were lowest, those of the rat intermediate, and those of mouse highest in activity for these enzymes. The enzyme levels did not vary as an exact power function of body weight, nor were there any intraspecific changes in enzyme levels with changes in body size.