Modulation of succinate dehydrogenase in response to environmental stress conditions of hypobaria and hypoxia

1. 1. An increase in the oxidation of succinate by hepatic mitochondria in rats exposed to hypoxia (O2-N2; 1:9, v/v) or hypobaria (0.5 atm) was observed which appears to be due to modification of the activity of the rate-limiting succinate dehydrogenase [succinate: (acceptor) oxidoreductase, EC 1.3.99.1].

Nature of the activation of succinate dehydrogenase byvarious effectors and in hypobaria and hypoxia

Hepatic mitochondrial succinate dehydrogenase (succinate:(acceptor)oxidoreductase, EC 1.3.99.1) was activated by preincubation of mitochondria with four diverse classes of compounds, the dicarboxylic acids, nitrophenols, quinols (and ubiquinols) and pyrophosphates. Of the various compounds tested malonate, oxaloacetate and pyrophosphate, well-known competitive inhibitors of the enzyme, and also hydroquinone and ubiquinols were effective even at low concentrations and showed maximal stimulation in 2 min.

Oxidation of succinate in heart, brain, and kidney mitochondria in hypobaria and hypoxia

Exposure of rats to hypobaric stress for periods of up to 36 h caused a consistent change in the succinate-NT reductase activity of the heart mitochondria whereas there was no significant change in the activities of either succinate dehydrogenase and succinate-NT reductase of the brain and the kidney. Mitochondrial succinate dehydrogenase of the heart, the brain and the kidney was activated 2- to 7-fold with the substrate and malonate. The activations obtained with oxalate, citrate and dinitrophenol were relatively lower in comparison to succinate and malonate. Benzohydroquinone and 2-nitrophenol had no stimulatory effect on the heart, the brain and the kidney mitochondria. THE ACTIVATIONS OBTAINED WITH THE VARIOUS EFFECTORS PARTIALLY (OR COMPLETELY IN THE CASE OF SUCCINATE) REVERSED ON WASHING THE MITOCHONDRIAL SAMPLES WITH THE SUCROSE HOMOGENIZING MEDIUM. The effect of ubiquinol, which also activated the enzyme, was only partially reversed after the second preincubation with succinate in the brain and the kidney whereas in the heart the activity was fully reversed. The increased activity of succinate dehydrogenase obtained with ATP and ADP was further enhanced by Mg2+ exclusively in the brain mitochondria, suggesting the possibility of Mg2+-AIP complex as the active species. Succinate-NT reductase of the heart, the brain and the kidney mitochondria showed a high activation with ubiquinone whereas its reduced form had no stimulatory effect.

An optimal dynamic inversion-based neuro-adaptive approach for treatment of chronic myelogenous leukemia

Combining the advanced techniques of optimal dynamic inversion and model-following neuro-adaptive control design, an innovative technique is presented to design an automatic drug administration strategy for effective treatment of chronic myelogenous leukemia (CML). A recently developed nonlinear mathematical model for cell dynamics is used to design the controller (medication dosage). First, a nominal controller is designed based on the principle of optimal dynamic inversion. This controller can treat the nominal model patients (patients who can be described by the mathematical model used here with the nominal parameter values) effectively. However, since the system parameters for a realistic model patient can be different from that of the nominal model patients, simulation studies for such patients indicate that the nominal controller is either inefficient or, worse, ineffective; i.e. the trajectory of the number of cancer cells either shows non-satisfactory transient behavior or it grows in an unstable manner. Hence, to make the drug dosage history more realistic and patient-specific, a model-following neuro-adaptive controller is augmented to the nominal controller. In this adaptive approach, a neural network trained online facilitates a new adaptive controller. The training process of the neural network is based on Lyapunov stability theory, which guarantees both stability of the cancer cell dynamics as well as boundedness of the network weights. From simulation studies, this adaptive control design approach is found to be very effective to treat the CML disease for realistic patients. Sufficient generality is retained in the mathematical developments so that the technique can be applied to other similar nonlinear control design problems as well.

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

Objectives: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. Design and methods: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated baernoglobin (HbA1c) was measured by HPLC. Results: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. Conclusions: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A neuro-adaptive augmented optimal dynamic inversion approach for effective and efficient treatment of chronic myelogenous leukemia

Combining the advanced techniques of optimal dynamic inversion and model-following neuro-adaptive control design, an efficient technique is presented for effective treatment of chronic myelogenous leukemia (CML). A recently developed nonlinear mathematical model for cell dynamics is used for the control (medication) synthesis. First, taking a set of nominal parameters, a nominal controller is designed based on the principle of optimal dynamic inversion. This controller can treat nominal patients (patients having same nominal parameters as used for the control design) effectively. However, since the parameters of an actual patient can be different from that of the ideal patient, to make the treatment strategy more effective and efficient, a model-following neuro-adaptive controller is augmented to the nominal controller. In this approach, a neural network trained online (based on Lyapunov stability theory) facilitates a new adaptive controller, computed online. From the simulation studies, this adaptive control design approach (treatment strategy) is found to be very effective to treat the CML disease for actual patients. Sufficient generality is retained in the theoretical developments in this paper, so that the techniques presented can be applied to other similar problem as well. Note that the technique presented is computationally non-intensive and all computations can be carried out online.

Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected Mycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata)

Administration of Tamoxifen via the Alzet pump at a rate of 50 mu g hr(-1) for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

Hypersensitivity of hypoxia grown Mycobacterium smegmatis to DNA damaging agents: Implications of the DNA repair deficiencies in attenuation of mycobacteria

Mycobacteria are an important group of pathogenic bacteria. We generated a series of DNA repair deficient strains of Mycobacterium smegmatis, a model organism, to understand the importance of various DNA repair proteins (UvrB, Ung, UdgB, MutY and Fpg) in survival of the pathogenic strains. Here, we compared tolerance of the M. smegmatis strains to genotoxic stress (ROS and RNI) under aerobic, hypoxic and recovery conditions of growth by monitoring their survival. We show an increased susceptibility of mycobacteria to genotoxic stress under hypoxia. UvrB deficiency led to high susceptibility of M. smegmatis to the DNA damaging agents. Ung was second in importance in strains with single deficiencies. Interestingly, we observed that while deficiency of UdgB had only a minor impact on the strain's susceptibility, its combination with Ung deficiency resulted in severe consequences on the strain's survival under genotoxic stress suggesting a strong interdependence of different DNA repair pathways in safeguarding genomic integrity. Our observations reinforce the possibility of targeting DNA repair processes in mycobacteria for therapeutic intervention during active growth and latency phase of the pathogen. High susceptibility of the UvrB, or the Ung/UdgB deficient strains to genotoxic stress may be exploited in generation of attenuated strains of mycobacteria. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

One-Carbon Metabolic Pathway Rewiring in Escherichia coli Reveals an Evolutionary Advantage of 10-Formyltetrahydrofolate Synthetase (Fhs) in Survival under Hypoxia

In cells, N-10-formyltetrahydrofolate (N-10-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N-10-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringens fhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (Delta folD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N-10-fTHF in the Delta folD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N-10-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The Delta folD strain (harboring fhs on the chromosome) showed a high NADP(+)-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria.