Influence of cerebral ischemia and post-ischemic reperfusion on mitochondrial oxidative phosphorylation

Unilateral ischemia in the right cerebral hemisphere of the rat was induced by ligation of the right common carotid artery coupled with controlled hemorrhage to produce hypotension (25±8 mm/Hg). Where indicated after 30 min of ischemia, the withdrawn blood was reinfused to restore arterial pressure to normal. Mitochondria isolated from the ipsilateral hemisphere after 30 min of ischemia showed significantly lower respiratory rates than the organelles isolated from the contralateral side. Oxidation of NAD+-linked substrates was more sensitive to inhibition in ischemia (30%) than was of ferrocytochromec (12%), succinate oxidation being intermediate. The activities of membrane-bound dehydrogenases (both NADH and succinate-linked) were also significantly lowered. Ischemia did not affect the cytochrome content of mitochondria. Respiratory activity (NAD+-linked) of mitochondria isolated from the ipsilateral hemisphere was twice as sensitive to inhibition by fatty acid as was of preparations from the contralateral side. Mitochondria isolated from cerebral cortex after 90 min of post-ischemic reperfusion showed no significant improvement in the rate of substrate oxidation. Adenine nucleotide translocase activity and energy-dependent Ca2+ uptake, both of which decreased significantly in mitochondria isolated from the ischemic brain, showed little recovery, on reperfusion. These observations suggested the strong possibility that the deleterious effects of ischemia on mitochondrial respiratory function might be mediated by free fatty acids that are known to accumulate in large amounts in ischemic tissues. The pattern of inhibition of ATPase activity was consistent with this view.

Major depression with ischemic heart disease: Effects of paroxetine and nortriptyline on measures of nonlinearity and chaos of heart rate

Depression is associated with increased cardiovascular mortality in patients with preexisting cardiac illness. A decrease in cardiac vagal function as suggested by a decrease in heart rate variability (HRV) or heart period variability has been linked to sudden death in patients with cardiac disease as well as in normal controls. Recent studies have shown decreased vagal function in cardiac patients with depression as well as in depressed patients without cardiac illness. In this study, we compared 20 h awake and sleep heart period nonlinear measures using quantification of nonlinearity and chaos in two groups of patients with major depression and ischemic heart disease (mean age 59-60 years) before and after 6 weeks of treatment with paroxetine or nortriptyline. Patients received paroxetine, 20-30 mg/day or nortriptyline targeted to 190-570 nmol/l for 6 weeks. For HRV analysis, 24 patients were included in the paroxetine treatment study and 20 patients in the nortriptyline study who had at least 20,000 s of awake data. The ages of these groups were 60.4 +/- 10.5 years for paroxetine and 60.8 +/- 13.4 years for nortriptyline. There was a significant decrease in the largest Lyapunov exponent (LLE) after treatment with nortriptyline but not paroxetine. There were also significant decreases in nonlinearity scores on S-netPR and S-netGS after nortriptyline, which may be due to a decrease in cardiac vagal modulation of HRV. S-netGS and awake LLE were the most significant variables that contributed to the discrimination of postparoxetine and postnortriptyline groups even with the inclusion of time and frequency domain measures. These findings suggest that nortriptyline decreases the measures of chaos probably through its stronger vagolytic effects on cardiac autonomic function compared with paroxetine, which is in agreement with previous clinical and preclinical reports. Nortriptyline was also associated with a significant decrease in nonlinearity scores, which may be due to anticholinergic and/or sympatholytic effects. As depression is associated with a strong risk factor for cardiovascular mortality, one should be careful about using any drug that adversely affects cardiac vagal function. Copyright (C) 2002 S. Karger AG, Basel.

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate

Copper(II) complexes Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H(2)satP) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido3,2-a:2',3'-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near -0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (similar to 1.85 mu(B)) are avid DNA binders giving K(b) values within 1.0 x 10(5) - 8.0 x 10(5) M(-1). Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC(50) = 8.3(+/- 1.0) mu M) in visible light, while showing lower dark toxicity (IC(50) = 17.2(+/- 1.0) mu M). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC(50) = 30.0(+/- 1.0) mu M in dark), while retaining its photocytotoxicity (IC(50) = 8.0(+/- 1.0) mu M). (C) 2011 Elsevier Inc. All rights reserved.

Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

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.

Neuroprotective activity of Wedelia calendulacea on cerebral ischemia/reperfusion induced oxidative stress in rats

Objective: This study was undertaken to evaluate the neuroprotective activity of Wedelia calendulacea against cerebral ischemia/reperfusion induced oxidative stress in the rats. Materials and Methods: The global cerebral ischemia was induced in male albino Wistar rats by occluding the bilateral carotid arteries for 30 min followed by 1 h and 4 h reperfusion. At various times of reperfusion, the histopathological changes and the levels of malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GST), and hydrogen peroxide (H(2)O(2)) activity and brain water content were measured. Results: The ischemic changes were preceded by increase in concentration of MDA, hydrogen peroxide and followed by decreased GPx, GR, and GST activity. Treatment with W. calendulacea significantly attenuated ischemia-induced oxidative stress. W. calendulacea administration markedly reversed and restored to near normal level in the groups pre-treated with methanolic extract (250 and 500 mg/kg, given orally in single and double dose/day for 10 days) in dose-dependent way. Similarly, W. calendulacea reversed the brain water content in the ischemia reperfusion animals. The neurodegenaration also conformed by the histopathological changes in the cerebral-ischemic animals. Conclusion: The findings from the present investigation reveal that W. calendulacea protects neurons from global cerebral-ischemic injury in rat by attenuating oxidative stress.

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.

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

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.

Metal Complexes of Curcumin for Cellular Imaging, Targeting, and Photoinduced Anticancer Activity

CONSPECTUS: Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-kappa B besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and damaging the cancer cells on photoactivation in visible light while being minimally toxic in darkness. In this Account, we have made an attempt to review the current status of the chemistry of metal curcumin complexes and present results from our recent studies on curcumin complexes showing remarkable in vitro photocytotoxicity. The undesirable dark toxicity of the complexes can be reduced with suitable choice of the metal and the ancillary ligands in a ternary structure. The complexes can be directed to specific subcellular organelles. Selectivity by targeting cancer cells over normal cells can be achieved with suitable ligand design. We expect that this methodology is likely to provide an impetus toward developing curcumin-based photochemotherapeutics for anticancer treatment and cure.

Quantitative metabolic profiling of NMR spectral signatures of branched chain amino acids in blood serum

Branched Chain Amino Acids (BCAAs) are related to different aspects of diseases like pathogenesis, diagnosis and even prognosis. While in some diseases, levels of all the BCAAs are perturbed; in some cases, perturbation occurs in one or two while the rest remain unaltered. In case of ischemic heart disease, there is an enhanced level of plasma leucine and isoleucine but valine level remains unaltered. In `Hypervalinemia', valine is elevated in serum and urine, but not leucine and isoleucine. Therefore, identification of these metabolites and profiling of individual BCAA in a quantitative manner in body-fluid like blood plasma/serum have long been in demand. H-1 NMR resonances of the BCAAs overlap with each other which complicates quantification of individual BCAAs. Further, the situation is limited by the overlap of broad resonances of lipoprotein with the resonances of BCAAs. The widely used commercially available kits cannot differentially estimate the BCAAs. Here, we have achieved proper identification and characterization of these BCAAs in serum in a quantitative manner employing a Nuclear Magnetic Resonance-based technique namely T-2-edited Correlation Spectroscopy (COSY). This approach can easily be extended to other body fluids like bile, follicular fluids, saliva, etc.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygenrich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.