Pharmacological inhibition of SIRT-2 by AK-7 modulates redox status and apoptosis via regulating Nrf2 in an experimental model of chronic obstructive pulmonary disease: an invivo and insilico study.

Chronic obstructive pulmonary disease (COPD) is defined by inflammation and emphysema. Sirtuins (SIRT) are NAD+-dependent histone deacetylases that regulate oxidative stress and inflammation. The present work investigates the modulatory role of SIRT-2 in experimental COPD model. Insilico comparative assessment of SIRT-2 inhibitors (AK-7 and AGK-2) by ADMET and molecular docking revealed AK-7 as suitable candidate for invivo application. COPD in mice was established by cigarette smoke (CS) exposure for 2 months. AK-7 (100 µg/kg and 200 µg/kg body weight) was administered intranasally one hour before CS exposure. The present investigation demonstrates that CS exposure increases total cell count, and free radical production (total reactive oxygen species, total oxidant status, myeloperoxidase, and nitric oxide), which were decreased by AK-7. It also altered antioxidant enzymatic activity (total antioxidant status, catalase, superoxide dismutase, glutathione peroxidase, glutathione-s-transferase, glutathione reductase, and reduced glutathione), hence preserving the redox balance. AK-7 significantly decreases apoptosis, protein carbonylation, lipid peroxidation, TNF-α and IFN-ï»» levels represent COPD generation in mice and were dramatically decreased by AK-7. Histopathological studies shows that CS exposure damages alveoli and produces peribronchiolar inflammation; both of these events were reduced by AK-7. The antioxidative potency of AK-7 was confirmed by observing Nrf2 and Keap1 proteins. Keap-dependent Nrf2 regulation was observed, with cytosolic Nrf2 and Keap1 expression elevated in COPD and reduced in the AK-7 group while nuclear Nrf2 was reduced in COPD and increased in the AK-7 group. The present study concludes that inhibition of SIRT-2 minimizes COPD severity and mediates therapeutic effects in the lungs.

Potential of hydroethanolic leaf extract of Ocimum sanctum in ameliorating redox status and lung injury in COPD: an in vivo and in silico study.

Oxidative stress and inflammation are hypothesised as the main contributor for Chronic Obstructive Pulmonary Disease (COPD). Cigarette smoke (CS), a major cause of COPD leads to inflammation resulting in recruitment of neutrophils and macrophages which are rich sources of oxidants. Activation of these cells produces excess oxidants and depletes antioxidants resulting in stress. Presently, effective drug for COPD is limited; therefore, novel compounds from natural sources, including plants are under exploration. The present study aims to investigate the protective effect of Ocimum sanctum leaf extract (OLE) in CS - induced model of COPD. Exposure to CS was performed thrice a week for 8 weeks and OLE (200 mg/kg and 400 mg/kg) was administered an hour before CS exposure. Control group (negative control) were exposed to ambient air while COPD group was exposed to CS (positive control). Administration of OLE doses reduced inflammation, decreased oxidant concentration and increased antioxidant concentration (p < 0.01). Molecular docking studies between the major phytocompounds of OLE (Eugenol, Cyclohexane and Caryophyllene) and antioxidant enzymes Superoxide dismutase (SOD), Catalase, Glutathione peroxidase (GPx), Glutathione reductase (GR) and Glutathione S Transferase (GST) showed strong binding interaction in terms of binding energy. In vivo and in silico findings for the first time indicates that OLE extract significantly alleviates oxidative stress by its potent free radical scavenging property and strong interaction with antioxidant enzymes. OLE extract may prove to be a therapeutic option for COPD prevention and treatment.

Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system.

Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 105 CFU, equating to 1.2 × 103 CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 101 CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays. Graphical abstract.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water.

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.

Aggravation of cyclophosphamide-induced reproductive toxicity in mice by aqueous extract of Aegle marmelos (L. )

ABSTRACT Aegle marmelos (L.) (Rutaceae) commonly known as bael is an important medicinal fruit tree. The present study focused on the effects of aqueous extract of Aegle marmelos (AEAM) on the testis and sperm characteristics induced by cyclophosphamide (CPA) in mice. Thirty six adult Parke's strain mice were divided into six groups: group I given only distilled water (control); group II administered with AEAM alone once in a week for five weeks; group III administered with CPA (200 mg/kg b.w., intraperitoneally) once in a week for five weeks and group IV-VI CPA along with AEAM (400, 500 and 600 mg/kg b.w., orally). CPA was found to reduce gonadosomatic index (GSI), sperm counts, motility, viability, antioxidant activities and induced histopathological changes of testis. In the group administered AEAM with CPA an exacerbation of sperm count, motility and viability of the cauda epididymis, GSI, antioxidant activities and architecture of testis was observed. The results suggest that the administration of AEAM may aggravate CPA-induced reproductive toxicity. It may be helpful in preparation of natural male contraceptives.

Antioxidant potential of Phyllanthus fraternus Webster on cyclophosphamide induced changes in sperm characteristics and testicular oxidative damage in mice.

Cyclophasphamide (CPA) is used to treat various types of cancer. It is a cytotoxic alkylating agent widely used in chemotherapeutic regimen. However, the clinical efficacy of CPA is marred by its side effects. In clinical applications of CPA, it becomes necessary to prevent the oxidative stress and reproductive toxicity induced thereby in normal cells. In the present study, we investigated the protective effect of aqueous extract of Phyllanthus fraternus (AEPF) on CPA (200 mg/kg body wt., i.p.) induced changes in sperm characteristics and testicular oxidative damage in male mice. The CPA treated group showed significant decrease in gonadosomatic index (GSI), epididymal sperm count, sperm motility and sperm viability compared to control group, while the CPA + AEPF treated group had significant increase with respect to these variables compared to the CPA-treated group. The elevated levels of lipid peroxidation by CPA were effectively reduced with AEPF. It also exhibited protective action against the CPA induced depletion of antioxidants like catalase and superoxide dismutase. DNA damage was measured by comet assay, biomonitoring with comet assay elicited significant increase in genotoxicity. Genotoxicity caused by CPA was counteracted by aqueous extract of Phyllanthus fraternus. Administration of the plant extract along with CPA restored the histopathological architecture of testis. Thus, the aqueous extract of P. fraternus by virtue of its antioxidant potential can be used as an effective agent to reduce CPA-induced oxidative stress in male mice.

Evaluation of the antioxidant and hepatoprotective effect of Phyllanthus fraternus against a chemotherapeutic drug cyclophosphamide.

In the present study, hepatoprotective and antioxidant properties of aqueous extract of Phyllanthus fraternus (AEPF 200, 300, and 400 mg/kg body weight (bw), orally) were investigated against cyclophosphamide (CPA 200 mg/kg, bw, intraperitoneally administered) induced liver damage in mice. Histopathological studies of CPA administration cause liver injury, featuring substantial increase in serum glutamic oxaloacetic transaminase, serum glutamate pyruvate transaminase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, and total bilirubin. Moreover, CPA intoxication also causes strong oxidative stress, which is evident from significant increase in lipid peroxide level. These changes were coupled with a decline in superoxide dismutase and catalase as well as albumin, cholesterol level, red blood cell (RBC), and white blood cell (WBC) count. AEPF-treated mice displayed a significant inhibition of lipid peroxidation and augmentation of endogenous antioxidants. The study emphasizes the hepatoprotective effect of AEPF against CPA-induced oxidative liver injury, which may serve as a promising medicinal herb in complementary chemotherapeutic modalities.

Cellular responses to misfolded proteins and protein aggregates.

Maintenance of the proteome is a major homeostatic task of the cell and disregulation of protein homeostasis can be deadly. The accumulation of different forms of misfolded protein can perturb protein homeostasis and cause extensive cell and tissue damage. The cell has various quality control systems to help prevent the accumulation of misfolded proteins and the complexity of the different mechanisms that have evolved is bewildering. The first order of business for all quality control systems is recognition of misfolded proteins, which is followed by a triage decision. In many cases, modular molecular chaperones function in different assemblies with degradatory or folding co-factors to direct a misfolded protein toward continued life or death. Herein, an overview of quality control mechanisms that triage soluble cytosolic proteins, protein aggregates, and ER-associated proteins is presented.

PLP-dependent H(2)S biogenesis.

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine ß-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Pre-steady-state kinetic analysis of enzyme-monitored turnover during cystathionine ß-synthase-catalyzed H(2)S generation.

Cystathionine ß-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals, i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H(2)S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457-22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H(2)S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H(2)S generation by yCBS is the ß-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H(2)S and forms an aminoacrylate intermediate (k(obs) of 1.61 ± 0.04 mM(-1) s(-1) at low cysteine concentrations and 2.8 ± 0.1 mM(-1) s(-1) at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM(-1) s(-1) and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products, cystathionine and H(2)S.

Investigations of low-frequency vibrational dynamics and ligand binding kinetics of cystathionine beta-synthase.

Vibrational coherence spectroscopy is used to study the low frequency dynamics of the truncated dimer of human cystathionine beta-synthase (CBS). CBS is a pyridoxal-5'-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. A strong correlation between the "detuned" coherence spectrum (which probes higher frequencies) and the Raman spectrum is demonstrated, and a rich pattern of modes below 200 cm(-1) is revealed. Normal coordinate structural decomposition (NSD) of the ferric CBS crystal structure predicts the enhancement of normal modes with significant heme "doming", "ruffling", and "saddling" content, and they are observed in the coherence spectra near approximately 40, approximately 60, and approximately 90 cm(-1). When pH is varied, the relative intensities and frequencies of the low frequency heme modes indicate the presence of a unique protein-induced heme structural perturbation near pH 7 that differs from what is observed at higher or lower pH. For ferric CBS, we observe a new mode near approximately 25 cm(-1), possibly involving the response of the protein, which exhibits a phase jump of approximately pi for excitation on the blue and red side of the Soret band maximum. The low frequency vibrational coherence spectrum of ferrous CBS is also presented, along with our efforts to probe its NO-bound complex. The CO geminate rebinding kinetics of CBS are similar to the CO-bound form of the gene activator protein CooA, but with the appearance of a significant additional kinetic inhomogeneity. Analysis of this inhomogeneity suggests that it arises from the two subunits of CBS and leads to a factor of approximately 20 for the ratio of the average CO geminate rebinding rates of the two subunits.

Heme regulation of human cystathionine beta-synthase activity: insights from fluorescence and Raman spectroscopy.

Cystathionine beta-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C horizontal lineN bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H2S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H2S generation. CBS from both organisms shows a marked preference for H2S generation by beta-replacement of cysteine by homocysteine. The alternative H2S-generating reactions, i.e. beta-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H2S production by CBS would account for approximately 25-70% of the total H2S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H2S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H2S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H2S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.

H2S biogenesis by human cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.

Although there is a growing recognition of the significance of hydrogen sulfide (H(2)S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H(2)S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine beta-synthase and cystathionine gamma-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H(2)S from cysteine and homocysteine. The gamma-replacement reaction, which condenses two molecules of homocysteine, yields H(2)S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a beta-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the alpha,beta-elimination of cysteine accounts for approximately 70% of H(2)S generation. However, the relative importance of homocysteine-derived H(2)S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 microm), the alpha,gamma-elimination and gamma-replacement reactions of homocysteine together are predicted to account for approximately 90% of H(2)S generation by CSE. Excessive H(2)S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.

Modulation of the heme electronic structure and cystathionine beta-synthase activity by second coordination sphere ligands: The role of heme ligand switching in redox regulation.

In humans, cystathionine beta-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is approximately 20A away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52, respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is -350+/-4mV and is substantially lower than the value of -287+/-2mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.