Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy

Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from −173 to +25°C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼10¹⁵ times slower at liquid nitrogen temperature than at room temperature.     

Observation of Two Inorganic Phosphate NMR Resonances in the Perfused Hypothermic Rat Heart

The effect of hypothermia on isolated perfused rat hearts was studied with³¹P NMR. Hearts were continuously perfused with phosphate-free Krebs–Henseleit buffer while the perfusate temperature was adjusted. Perfusate pH was kept at 7.40 ± 0.02 throughout the experiments. Using the chemical shift difference between PCr and P_ithe intracellular pH was estimated. At 36, 20, and 10°C a cytosolic alkalinization at a pH of 7.05 ± 0.04, 7.21 ± 0.05, and 7.40 ± 0.03 was observed, respectively. At 10°C two P_iresonances were observed with a separation of 0.25 ppm. This resonance corresponded to a P_iresonance of a cellular compartment with a local pH of 7.78 ± 0.06, likely mitochondrial. This additional resonance disappeared upon warming of the hearts back to 36°C.     

Biogenesis of Mitochondria in Germinating Peanut Cotyledons II. Changes in Cytochromes and Mitochondrial DNA

Biogenesis of mitochondria occurs in the germinating cotyledons of peanuts. This process was demonstrated by measuring both constitutive and enzymatic properties of mitochondria as a function of germination time. Direct counting by phase contrast microscopy of sucrose density gradient preparations showed that the number of mitochondria increased markedly during germination. DNA with a buoyant density distinct from the major cellular DNA was associated with these mitochondrial preparations. During germination the amount of this DNA in mitochondrial pellets increased. This increase closely paralleled the increase in number of mitochondria.

Succinoxidase and succinic dehydrogenase increased during germination. Both activities were confined to the mitochondrial fraction. The rate of increase of succinoxidase activity was significantly greater than the rate of increase of succinic dehydrogenase and both increased at least initially at a greater rate than the amount of mitochondrial DNA or numbers of mitochondria.

The amounts of cytochromes present in mitochondrial preparations were measured spectrophotometrically. All of the cytochromes increased in amount during germination. The rate of increase of cytochrome a — a₃ very close to the rate of increase in succinoxidase activity.

     

In Situ Characterization of Renal Insulin Receptrors in the Rat

Abstract

Insulin regulates carbohydrate metabolism, and water, sodium, potassium, and phosphate reabsorption in the kidney by binding to specific receptors. Insulin receptors have been identified in the kidney using membrane preparations obtained from both glomeruli and tubules. In this study, an autoradiographic technique was used to characterize insulin receptors in the rat kidney. Frozen tissue sections were preincubated to remove endogenously bound insulin, incubated in a buffer containing 200 pM 12sl-Tyr-insulin, washed, and dried before exposure on Ultrofilm. Binding density was assessed by computerized microdensitometry. In the cortex, binding density was comparable in glomeruli and tubules. In the medulla, bound radioligand was found primarily in longitudinal structures traversing the outer portion, presumably corresponding to vascular bundles, and in the inner portion. Scatchard analysis of competition binding data resulted in curvilinear profiles, indicating either two classes of receptors with different affinity or the presence of a single class of receptors with a negative cooperative hormone-receptor interaction. Data analyzed for a two-site model showed one receptor site with Kd of 0.39f0.14 nmol/l and Bmax of 3. M. O × 1010 receptordmma and another site with Kd of 0.30fl.1 pmoVl and a Bmax of 3.2 × 1013 receptordmms. Thus, in situ autoradiography can be used to determine distribution and binding characteristics of insulin receptors in rat kidney and might be employed in receptor studies on rat models of human disease.     

In Vitro and In Situ Absorption of Methionine Sulfoxide in Rat Small Intestine

Absorption of methionine and its sulfoxide was investigated in vitro with everted sacs and in situ with circulated loops of rat small intestine. Transmural transport and tissue accumulation of methionine sulfoxide in the everted sacs were in fair agreement with those of methionine. Apparent kinetic parameters for the difference of transmural transport in the absence and presence of 10^?5 m carbonylcyanide m-chlorophenylhydrazone, i.e. for the energy-dependent active transport, were similar for both methionine and its sulfoxide. Methionine was found at a low level in the serosal fluid of the everted sac on incubation with methionine sulfoxide. It was attributed to the methionine leaked out from the tissue but not to that formed by reduction of methionine sulfoxide during the course of intestinal transport. Similar transport was also observed in situ in circulated intestinal loops for methionine and its sulfoxide. The absorption efficiency of methionine sulfoxide in the small intestine is not the reason for the decreased nutritional availability of the most likely oxidation product of methionine.     

Changes in NMDAR2 Subunit mRNA Levels During Pentobarbital Tolerance/Withdrawal in the Rat Brain: An In Situ Hybridization Study

Little is known about the functional modulation of NMDA receptor subunits at the molecular level. Therefore, a series of experiments were conducted to elucidate more fully the role of NMDA receptor subtypes in pentobarbital tolerance and withdrawal. We investigated the influence of centrally administered pentobarbital on the regulation of mRNA levels of the family of NMDA receptor 2 (NR2) subtypes (NR2A, NR2B, and NR2C) by in situ hybridization histochemistry in rat brain. Animals were rendered tolerant by continuous intracerebroventricular (i.c.v.) infusion with pentobarbital (300 g/10 l/hr for 6 days) through pre-implanted cannulae connected to osmotic mini-pumps, and dependent, by abrupt withdrawal from pentobarbital. The NR2A subunit mRNA was increased in cortical areas in pentobarbital tolerant and withdrawal rats. In contrast, the NR2B mRNA was decreased in parietal cortex and hippocampus in both tolerance and withdrawal rats. The level of NR2C mRNA was increased in withdrawal rats, while there was no change in tolerant rats. These results indicate that continuous i.c.v. infusion with pentobarbital alters NR2 subunit mRNA expression in the rat brain, suggesting that NR2 subunits may play an important role in the development of tolerance to and withdrawal from pentobarbital.     

耗竭性游泳对大鼠心肌线粒体膜功能的影响

耗竭性游泳对大鼠心肌线粒体膜功能的影响王文信,丁树哲,许豪文（华东师范大学体育系运动生化实验室,上海２０００６２）关键词心肌线粒体,内膜表面电位,游离钙,总钙,合成活力长时间耗竭性游泳或跑步引起心肌、骨骼肌、肝脏等线粒体结构和功能变化,这些变化可能与...     

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.     

Bifurcations and Intrinsic Chaotic and 1/f Dynamics in an Isolated Perfused Rat Heart

The application of the theory of chaotic dynamical systems has gradually evolved from computer simulations to assessment of erratic behavior of physical, chemical, and biological systems. Whereas physical and chemical systems lend themselves to fairly good experimental control, biologic systems, because of their inherent complexity, are limited in this respect. This has not, however, prevented a number of investigators from attempting to understand many biologic periodicities. This has been especially true regarding cardiac dynamics: the spontaneous beating of coupled and non-coupled cardiac pacemakers provides a convenient comparison to the dynamics of oscillating systems of the physical sciences. One potentially important hypothesis regarding cardiac dynamics put forth by Goldberger and colleagues, is that normal heart beat fluctuations are chaotic, and are characterized by a 1/f-like power spectrum. To evaluate these conjectures, we studied the heart beat intervals (R wave toR wave of the electocardiogram) of isolated, perfused rat hearts and their response to a variety of external perturbations. The results indicate bifurcations between complex patterns, states with positive dynamical entropies, and low values of fractal dimensions frequently seen in physical, chemical and cellular systems, as well as power law scaling of the spectrum. Additionally, these dynamics can be modeled by a simple, discrete map, which has been used to describe the dynamics of the Belousov-Zhabotinsky reaction.     

Intermittent Fasting Results in Tissue-Specific Changes in Bioenergetics and Redox State

Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart.     

Establishment of a Rat Model of Portal Vein Ligation Combined with In Situ Splitting

Background

Portal vein ligation (PVL) combined with in situ splitting (ISS) has been shown to induce remarkable liver regeneration in patients. The purpose of this study was to establish a model of PVL+ISS in rats for exploring the possible mechanisms of liver regeneration using these techniques.

Materials and Methods

Rats were randomly assigned to three experimental groups: selective PVL, selective PVL+ISS and sham operation. The hepatic regeneration rate (HRR), Ki-67, liver biochemical determinations and histopathology were assessed at 24, 48, and 72 h and 7 days after the operation. The microcirculation of the median lobes before and after ISS was examined by laser speckle contrast imaging. Meanwhile, cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes at 24 h was investigated by RT-PCR and ELISA.

Results

The HRR of PVL+ISS was much higher than that of the PVL at 72 h and 7 days after surgery (p<0.01). The expression of Ki-67 in hepatocytes in the regenerating liver lobe was stronger in the PVL+ISS group than in the PVL group at 48 and 72 h (p<0.01). There was a significant reduction in microcirculation blood perfusion of the left median lobe before and after ISS. Liver biochemical determinations and histopathology demonstrated more severe hepatocyte injury in the PVL+ISS group. Both the mRNA levels of TNF-α and IL-6 and the protein levels of TNF-α, IL-6 and HGF in regenerating liver lobes were higher in the PVL+ISS than the PVL alone.

Conclusions

The higher HRR in the PVL+ISS compared with the PVL confirmed that we had successfully established a PVL+ISS model in rats. The possible mechanisms included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines in the regenerating lobes after ISS.     

Redox State and Mitochondrial Respiratory Chain Function in Skeletal Muscle of LGMD2A Patients

Background

Calpain-3 deficiency causes oxidative and nitrosative stress-induced damage in skeletal muscle of LGMD2A patients, but mitochondrial respiratory chain function and anti-oxidant levels have not been systematically assessed in this clinical population previously.

Methods

We identified 14 patients with phenotypes consistent with LGMD2A and performed CAPN3 gene sequencing, CAPN3 expression/autolysis measurements, and in silico predictions of pathogenicity. Oxidative damage, anti-oxidant capacity, and mitochondrial enzyme activities were determined in a subset of muscle biopsies.

Results

Twenty-one disease-causing variants were detected along the entire CAPN3 gene, five of which were novel (c.338 T>C, c.500 T>C, c.1525-1 G>T, c.2115+4 T>G, c.2366 T>A). Protein- and mRNA-based tests confirmed in silico predictions and the clinical diagnosis in 75% of patients. Reductions in antioxidant defense mechanisms (SOD-1 and NRF-2, but not SOD-2), coupled with increased lipid peroxidation and protein ubiquitination, were observed in calpain-3 deficient muscle, indicating a redox imbalance primarily affecting non-mitochondrial compartments. Although ATP synthase levels were significantly lower in LGMD2A patients, citrate synthase, cytochrome c oxidase, and complex I+III activities were not different from controls.

Conclusions

Despite significant oxidative damage and redox imbalance in cytosolic/myofibrillar compartments, mitochondrial respiratory chain function is largely maintained in skeletal muscle of LGMD2A patients.     

Slow Penetration of Thyrotropin-Releasing Hormone Across the Blood-Brain Barrier of an In Situ Perfused Guinea Pig Brain

Transport of 3H-labelled thyrotropin-releasing hormone (TRH) across the blood-brain barrier was studied in the ipsilateral perfused in situ guinea pig forebrain. The unidirectional transfer constant (Kin) calculated from the multiple time brain uptake analysis ranged from 1.14 X 10(-3) to 1.22 X 10(-3) ml min-1 g-1, in the parietal cortex, caudate nucleus, and hippocampus. Regional Kin values for [3H]TRH were significantly reduced by 43-48% in the presence of an aminopeptidase and amidase inhibitor, 2 mM bacitracin, suggesting an enzymatic degradation of tripeptide during interaction with the blood-brain barrier. In the presence of unlabelled 1 mM TRH and 2 mM bacitracin together, a reduction of [3H]TRH regional Kin values similar to that obtained with 2 mM bacitracin alone was obtained . L-Prolinamide, the N-terminal residue of tripeptide, at a 10 mM level had no effect on the kinetics of entry of [3H]TRH into the brain. The data indicate an absence of a specific saturable transport mechanism for TRH presented to the luminal side of the blood-brain barrier. It is concluded that intact TRH molecule may slowly penetrate the blood-brain barrier, the rate of transfer being some three times higher than that of D-mannitol.     

Dopaminergic Regulation of Cannabinoid Receptor mRNA Levels in the Rat Caudate-Plitamen: An In Situ Hybridization Study

Abstract: By quantitative in situ hybridization, we examined in vivo in the rat caudate-putamen the effects on levels of cannabinoid receptor mRNA of an interruption of dopamine neurotransmission for up to 1 month, by either 6-hydroxydopamine lesioning of the medial forebrain bundle or dopamine receptor blockade. We found, in a first set of experiments, that unilateral 6-hydroxydopamine dopa-minergic deafferentation of the striatum (characterized by a contralateral turning behavior in response to apomor-phine, the almost complete disappearance of the tyrosine hydroxylase hybridization signal in the substantia nigra, and an increase of preproenkephalin A mRNA level in the striatum) was associated with significantly increased (45%) cannabinoid receptor mRNA levels in the homolateral caudate-putamen. In a second set of experiments, treatments with the dopamine D₁ receptor antagonist SCH-23390, haloperidol, and the D₂ receptor antagonist sulpiride induced significantly higher cannabinoid receptor mRNA levels (respectively, 67, 34, and 27%) in the caudate-putamen. These observations suggest for the first time that, in vivo, cannabinoid receptor gene expression in the caudate-putamen is under the negative control of dopamine receptor-mediated events.     

Activation of Integrins by Urea in Perfused Rat Liver

High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K⁺ channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552–560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of β₁ integrins followed by an activation of focal adhesion kinase, c-Src, p38^MAPK, extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α₅β₁ integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K⁺ efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38^MAPK inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α₅β₁ integrin in water, urea, or thiourea solutions revealed significant conformational changes of α₅β₁ integrin in urea and thiourea solutions, in contrast to the simulation of α₅β₁ in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α₅β₁ integrin system.     

The Extracellular Redox State Modulates Mitochondrial Function,Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.