Reduction Of Energy Charge Research Articles

Mini Bubble Columns for Miniaturizing Scale‐Down

ABSTRACTThe successful scale‐up of biotechnological processes from laboratory to industrial scale is crucial for translating innovation to practice. Scale‐down simulators have emerged as indispensable tools in this endeavor, enabling the evaluation of potential hosts’ adaptability to the dynamic conditions encountered in large‐scale fermenters. By simulating these real‐world scenarios, scale‐down simulators facilitate more accurate estimations of host productivity, thereby improving the process of selecting optimal strains for industrial production. Conventional scale‐down systems for detailed intracellular analysis necessitate an elaborate setup comprising interconnected lab‐scale reactors such as stirred tank reactors (STRs) and plug‐flow reactors (PFRs), often proving time‐consuming and resource‐intensive. This work introduces a miniaturized bubble column reactor setup (60 mL working volume), enabling individual and parallel carbon‐limited chemostat fermentations, offering a more efficient and streamlined approach. The industrially relevant organism Escherichia coli, chosen as a model organism, is continuously grown and subjected to carbon starvation for 150 s, followed by a return to carbon excess for another 150 s. The cellular response is characterized by the accumulation of the alarmone guanosine pentaphosphate (ppGpp) accompanied by a significant reduction in energy charge, from 0.8 to 0.7, which is rapidly replenished upon reintroduction of carbon availability. Transcriptomic analysis reveals a two‐phase response pattern, with over 200 genes upregulated and downregulated. The initial phase is dominated by the CRP–cAMP‐ and ppGpp‐mediated response to carbon limitation, followed by a shift to stationary phase‐inducing gene expression under the control of stress sigma factors. The system's validity is confirmed through a thorough comparison with a conventional STR/PFR setup. The analysis reveals the potential of the system to effectively reproduce data gathered from conventional STR/PFR setups, showcasing its potential use as a scale‐down simulator integrated in the process of strain development.

CPPU-induced changes in energy status and respiration metabolism of grape young berry development in relation to Berry setting

The energy status, the level of respiration metabolism are improtant index reflecting plant growth state, participates in many growth processes. However, their relationship to berry setting remain unclear. In this study, the grape of ‘kyoho’ of CPPU [CPPU, N-(2-coloro-4-pyridinyl)-N-phenylurea] treated were used to measurement physiological indicators. The results showed that CPPU treatment reduced respiratory rate, reduced Cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO) activity, reduced NAD kinase (NADK) activity, maintained low nicotinamide adenine denucleotie phosphate (NADP) and nicotinamide adenine denucleotie phosphate reduced form (NADPH) levels, reduced nicotinamide adenine denucleotie (NADH) levels, and slowed down the reduction of energy charge. These results provided strong evidence that CPPU treatment promoted grape berry setting due to increased energy status, increased Embden-Meyerhof pathway (EMP) and the tricarboxylic acid (TCA) cycle respiratory metabolic pathways, decreased pentose phosphate pathway (PPP), and decreased CCO and AAO activity of respiratory terminal oxidase.

Energy Metabolic Changes in the Early Post-injury Period Following Traumatic Brain Injury in Rats

Impaired cerebral energy metabolism may be a major contributor to the secondary injury cascade that occurs following traumatic brain injury (TBI). To estimate the cortical energy metabolic state following mild and severe controlled cortical contusion (CCC) TBI in rats, ipsi-and contralateral cortical tissues were frozen in situ at 15 and 40 min post-injury and adenylate (ATP, ADP, AMP) levels were analyzed using high-performance liquid chromatography (HPLC) and the energy charge (EC) was calculated. At 15 min post-injury, mildly brain-injured animals showed a 43% decrease in cortical ATP levels and a 2.4-fold increase in AMP levels (P < 0.05), and there was a significant reduction of the ipsilateral cortical EC when compared to sham-injured animals (P < 0.05). At 40 min post-injury, the ipsilateral adenylate levels and EC had recovered to the values observed in the sham-injury group. In the severe CCC group, there was a 51% decrease in ipsilateral cortical ATP levels and a 5.3-fold increase in AMP levels with a significant reduction of cortical EC at 15 min post-injury (P < 0.05). At 40 min post-injury, a 2.6-fold ipsilateral increase in AMP levels and an 11% and 44% decrease in EC and ATP levels, respectively, remained (P < 0.05). A 37-38% reduction of the total adenylate pool was observed ipsilaterally in both CCC severity groups at the early time-point, and a 19% and 28% decrease remained in the mild and severe CCC groups, respectively, at 40 min post-injury. Significant contralateral ATP and EC changes were only observed in the severe CCC group at 40 min post-injury (P < 0.05). The energy-requiring secondary injury cascades that occur early post-injury do not challenge the brain tissue to the extent of ATP depletion and may provide a window of opportunity for therapeutic intervention.

Skeletal muscle abnormalities in rats with experimentally induced heart hypertrophy and failure.

In congestive heart failure (CHF), function and metabolism of skeletal muscles are abnormal. To evaluate whether the reduced oxidative capacity of skeletal muscles in CHF is due to impaired O(2) utilisation. CHF was induced in rats by injecting 50 mg/Kg monocrotaline. Several animals received the same dose of monocrotaline but only compensated right ventricular hypertrophy and no sign of congestion resulted. Two age- and diet-matched groups of control animals were also studied. In soleus and extensor digitorum longus (EDL) muscles, we studied skeletal muscle blood flow, oxidative capacity and respiratory function of skinned muscle fibres. In CHF, we observed a decrease of muscle blood flow (statistically significant in the soleus, p < 0.05 vs. controls). In compensated rats, a similar trend in blood flow was observed. In both soleus and EDL, a significant reduction of high energy phosphate and a shift of the redox potential towards accumulation of reducing equivalents were observed. The reduction of energy charge was not correlated to the decrease of blood flow. In skinned myofibres, the ratio of O(2) utilised in the presence and in absence of ADP (an index of phoshorilating efficiency) was reduced from 8.9 +/- 1.9 to 2.7 +/- 0.2 (p < 0.001) and from 5.7 +/- 1.0 to 2.0 +/- 0.3 (p < 0.01) in soleus and EDL, respectively. Activity of the different complexes of respiratory chain was investigated by means of specific inhibitors, showing major abnormalities at the level of complex I. In fact, inhibition of VO(2) by rotenone was decreased from 83.5 +/- 3.2 to 36.4 +/- 9.6 % (p < 0.005) and from 81.8 +/- 6.1 to 38.2 +/- 7.4 % (p < 0.005) in soleus and EDL, respectively. In rats with CHF, abnormalities of oxidative phosphorylation of muscles occur and complex I of the respiratory chain seem to be primarily affected. The metabolic alterations of skeletal muscles in CHF may be explained, at least in part, by an impaired O(2) utilisation.

Beneficial Effects of the 21-Aminosteroid U 74389G on the Ischemia-reperfusion Damage in Pig Hearts

21-Aminosteroids (Lazaroids), acting as free radical scavengers and as membrane stabilizers, proved to have beneficial effects in various pathological conditions. In the present study we explored the effectiveness of one of these compounds, U 74389 G, in protecting pigs myocardium against the ischemia-reperfusion damage induced by transient coronary occlusion achieved by clampling the left anterior descending coronary artery. Animals were divided into three groups: control, untreated and treated. Control animals were operated but not subjected to ischemia-reperfusion; the untreated group underwent to ischemia-reperfusion without pharmacological treatment; while the treated group received the aminosteroid (4 mg/kg) before coronary occlusion and at the time of reperfusion. Specimens of myocardial tissue and blood samples were taken for morphological and biochemical studies. In the ischemic-reperfused myocardium of the untreated animals, the dominant morphological features were neutrophil infiltration, intercellular edema and severe swelling of mithocondria. All these alterations, notably neutrophil infiltration, were attenuated by aminosteroid treatment. As for the biochemical findings, the changes in adenine nucleotides and nucleosides levels, thus the reduction of energy charge, were reversed in the treated, but not in the untreated group. Myocardial concentration of malondialdehyde, which was undetectable in the control group, was raised in all the animals after reperfusion, but this effect was significantly less marked with aminosteroid treatment. In addition, the higher myocardial content of ascorbic acid and the reduced serum potential peroxidation exhibited by the treated animals compared with untreated group indicate an enhanced antioxidant protection induced by aminosteroid administration. On the other hand, the serum levels of myoglobin, cardiac troponin I and creatine kinase MB isoenzyme suggest the ability of the aminosteroid to attenuate the modifications of membrane permeability induced by ischemia-reperfusion injury. All these results lead to the conclusion that aminosteroid treatment, at least in the conditions of the present study, is effective in reducing the morphological and biochemical alterations occurring in ischemic-reperfused myocardium.

Metabolic responses to anoxia and freezing by the freeze tolerant marine mussel Geukensia demissus

The effects of 2 or 12 h freezing at −6 °C or anoxia exposure at 5 °C on the metabolic responses of gill, mantle, adductor muscle, and hepatopancreas were characterized in the freeze tolerant, intertidal ribbed mussel Geukensia demissus (Dillwyn). In general, freezing and anoxia elicited different metabolic responses and these also differed in a tissue-specific manner. Fermentative end products accumulated in all tissues except gill under both stresses. In adductor muscle, lactate was produced in both cases. In mantle and hepatopancreas, only succinate accumulated in anoxia, correlated with an opposite decrease in aspartate, and changes in glycolytic intermediates were consistent with anoxia-induced metabolic arrest. During freezing, however, both lactate and succinate accumulated (as well as alanine in hepatopancreas) with net end product accumulation 3–4-fold greater than during anoxia and changes in glycolytic intermediates were consistent with the derivation of these products from carbohydrate catabolism. Other differences in response to freezing versus anoxia included the accumulation of glucose in all tissues and a reduction of energy charge in mantle during freezing, but not in anoxia. Taurine and glycine comprised about 80–85% of the total free amino acid pools in all tissues; in gill levels of both amino acids fell significantly during freezing or anoxia exposures whereas in mantle a significant decrease in these amino acids occurred during anoxia only. The data show both a broader range of metabolic responses, and greater net changes in metabolite levels during freezing compared with anoxia. This suggests (1) that metabolic responses to freezing include both cryoprotective and ischemia-protective changes, and (2) that tissue mechanisms of metabolic rate depression are rapidly induced during anoxia exposure but develop more slowly during freezing.

Effects of the ionophore gramicidin D on energy metabolism in human erythrocytes.

Washed human erythrocytes were incubated in phosphate-buffered saline at 37 degrees C with different concentrations (2-80 mg l-1) of the monovalent cationophore gramicidin D. The ionophore induced a decrease of ATP content and energy charge (EC) and a concomitant increase in ADP and AMP contents. At a concentration of 10 mg l-1 of the ionophore the reduction of EC and ATP concentration was about 50%. Increasing the concentration of the ionophore resulted in a substantial haemolysis with no further effect on the nucleotide levels. Lowering the concentration of the ionophore to 5 mg l-1 (to avoid haemolysis) lead to a similar effect on the energy status. Ouabain at 0.5 mmol l-1 in incubation medium distinctly blunted the ionophoric effect on EC and adenine nucleotides. Addition of glucose on the contrary reversed these effects by about 80%. Hence, the glucose-induced increase in glycolytic flux as evidenced by an increase in lactate production was not sufficient to restore energy state.

Mechanism of peroxide‐induced cellular injury in cultured adult cardiac myocytes

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.

HUMAN TROPHOBLAST - SENSITIVITY TO GLUCOSE AND OXYGEN DEPRIVATION

Top of pageAbstract Trophoblast uses large amounts of glucose and oxygen to meet the energy requirements of its synthetic and transport functions. We studied the effects of glucose and oxygen shortage on energy metabolism in a highly enriched population of trophoblastic cells from normal placentae, obtained with collagenase digestion and density gradient centrifugation. Intracellular adenylate (ATP, ADP, AMP) pool was labeled with 14C-adenine. Metabolic integrity was monitored with the adenylate energy charge (EC). Glucose-free medium or 100% N2-atmosphere had no effect on EC. Inhibition of glycolysis with 2-deoxy-D-glucose (DG) caused a rapid reduction in adenylates to less than a fourth and EC to about 65% in both first and third trimester cultures. Inhibition of oxidative phosphorylation with rotenone (R) caused a slower reduction in EC (p<0.01) and in adenylates than DG in first trimester. The effect of R on EC was also less (p<0.01) in first than third trimester. R in the presence of glucose had no effect. Concl.: 1) the trophoblast is able to maintain its energy supply by glycolysis or respiration alone, 2) inhibition of ATP-production causes a profound deterioration in energy status, 3) exogenous glucose is required for glycolytic ATP-production, 4) susceptibility to hypoxia increases as a function of gestational age.

Effect of a failure of energy supply on adenine nucleotide breakdown in placentae and other fetal tissues from rat and guinea pig

The effects of ischaemia on adenylate energy charge of tissues from fetal rats and fetal guinea pigs were measured. Adult rat and guinea-pig tissues, as well as human placentae, were also studied. The largest differences observed were between the fetuses from different pregnant animals (P = 4.74 X 10(-15). Reductions in energy charge in placentae were slower than in other defined fetal tissues, especially brain. In the rat, an immature species at birth, greater 'stability' was observed in placentae of 14 days of gestation than near term at 20 days of gestation. As contrast, in the guinea pig, a mature species at birth, there was no difference in 'stability' in placenta or other fetal tissues between about 40 days of gestation and near term, about 60 days of gestation. In addition to these tissue and maturity effects in the fetus, it has been confirmed that fetal tissues are more 'resistant' than adult tissues to failures of energy supply. Concentrations of adenosine, uridine, guanosine and cytidine nucleotides in placenta show similar patterns in rats and guinea pigs. Fetal liver contains more uridine nucleotides and brain more cytidine nucleotides. It is suggested that the placenta retains an early fetal ability to maintain itself during ischaemia; this might be advantageous during parturition. Possible endocrine and other mechanisms 'damping' fetoplacental metabolism are linked with a discussion of the large maternal effect.

Metabolism of adenine nucleotides in the cultured fetal mouse heart.

Intact beating fetal mouse hearts in organ culture were deprived of oxygen and glucose for up to 4 h, resulting in loss of beating, an 80% fall in ATP, reduction of energy charge from 0.85 to 0.48, and doubling of total nucleoside concentration. Radiolabeled adenine nucleotides were degraded to hypoxanthine and inosine, which were lost from the hearts into the medium during the deprivation period. Adenosine and adenine also appeared in the medium when adenosine deaminase was inhibited. After 24 h of O2 and glucose resupply, ATP returned to 60% of control, and energy charge rose to 0.76. Labeled nucleosides and bases remaining in the heart or exogenous labeled adenine were utilized to resynthesize ATP. [14C]glycine was rapidly taken up by recovering hearts but was not used for de novo adenine nucleotide synthesis. Ability to recover ATP and spontaneous contraction appear related to residual nucleotide and nucleoside content rather than to energy charge.