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Zinc oxide nanoparticles (ZnO NP) are commonly used engineered NPs with extensive usage in consumer products, thus leading to direct exposure to humans. The direct route of exposure is through inhalation. Once inhaled, these particles accumulate in the lungs, increasing the chances of respiratory tract illness through cellular organelle damage. Zinc oxide nanoparticle-treated lung cells are reported to display cytotoxicity, increase DNA damage, and induce oxidative stress. The current study focused on the effects of ZnO NPs on mitochondrial dynamics (fission and fusion) in human lung epithelial cells (A549). The lung cells were exposed to ZnO NPs at 50 and 100 μg/ml concentrations, and their mitochondrial dynamics were assessed to understand the effects of the NPs. Treatment with ZnO NPs reduced the activity of mitochondrial complex I and complex III and altered mitochondrial structural and functional characteristics in a concentration-dependent manner. Zinc oxide nanoparticles exposure showed an increase in small and round-shaped mitochondria. The expression of various fission proteins (Drp1 and Fis1) and fusion proteins (Mfn1, Mfn2, and OPA1) was altered upon exposure to ZnO NPs. Our studies showed dysfunction of the mitochondria induced by ZnO NPs. In fibroblast mitochondrial dynamics, fission symbolizes threshold damage. In this paper, we have shown that the mitochondrial fission phenotype increased upon exposure to ZnO NPs. The paper emphasizes that these particles enter mitochondria, triggering a stress response that results in the removal of mitochondria via fission. It provides relevant data for safety guidelines to ensure the safer use of these particles.

The present study is conducted to understand the association of mood profile with the kynurenine pathway (KP) metabolites, and cerebral hemodynamics in freshly recruited central armed forces personnel. Profile of Mood States questionnaire was utilized to assess mood profile, and Total Mood Disturbance (TMD) score was calculated. Transcranial Doppler was used to record blood flow velocity bilaterally of the middle cerebral artery. Chromatographic profile of the kynurenine metabolites was obtained in serum. Further, personnel were stratified according to sociodemographic variables (gender, age and diet) to observe the changes in their KP metabolic status. An activation of the kynurenic acid branch of the KP and the reduction in the mean blood flow velocity, and an increase in Gosling pulsatility index (PI) were observed in females having high TMD score. On gender comparative analysis, kynurenine metabolites of quinolinic acid branch and serotonin were significantly high in males. In males, with increase in age, a significant increase in the quinolinic acid branch of the KP was observed. Furthermore, a significant difference in level metabolites of the KP among the vegetarian and non-vegetarian groups was also observed. In conclusion we observed that increased TMD score was associated with cerebral hypoperfusion and higher vascular resistance along with activation of the KP. Our findings highlighted the importance of multi-facet brain function to showcase the close interaction of various dimensionalities and true picture of the assessee.

The therapeutic efficiency of anticancer nucleoside analogues (NA) strongly depends on their intracellular accumulation and conversion into 5′-triphosphates. Because active NATP cannot be directly administrated due to instability, we present here a strategy of nanoencapsulation of these active drugs for efficient delivery to tumors. Stable lyophilized formulations of 5′-triphosphates of cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP) encapsulated in biodegradable PEG-cl-PEI or F127-cl-PEI nanogel networks (NGC and NGM, respectively) were prepared by a self-assembly procedure. Cellular penetration, in vitro cytotoxicity, and drug-induced cell cycle perturbations of these nanoformulations were analyzed in breast and colorectal cancer cell lines. Cellular accumulation and NATP release from nanogel was studied by confocal microscopy and direct high-performance liquid chromatography analysis of cellular lysates. Antiproliferative effect of dFdCTP nanoformulations was evaluated in human breast carcinoma MCF7 xenograft animal model. Nanoencapsulated araCTP, dFdCTP, and FdUTP showed similar to NA cytotoxicity and cell cycle perturbations. Nanogels without drugs showed very low cytotoxicity, although NGM was more toxic than NGC. Treatment by NATP nanoformulations induced fast increase of free intracellular drug concentration. In human breast carcinoma MCF7 xenograft animal model, i.v. dFdCTP-nanogel was equally effective in inhibiting tumor growth at four times lower administered drug dose compared with free gemcitabine. Active triphosphates of NA encapsulated in nanogels exhibit similar cytotoxicity and cell cycle perturbations in vitro and faster cell accumulation and equal tumor growth-inhibitory activity in vivo at much lower dose compared with parental drugs, illustrating their therapeutic potential for cancer chemotherapy. [Mol Cancer Ther 2008;7(10):3373–80]

Supplementary Fig. from Polymeric nanogels containing the triphosphate form of cytotoxic nucleoside analogues show antitumor activity against breast and colorectal cancer cell lines

Graphene quantum dots (GQDs) are a luminescent class of carbon nanomaterials with a graphene-like core structure, possessing quantum confinement and edge effects. They have gained importance in the biological world due to their inherent biocompatibility, good water dispersibility, excellent fluorescence and photostability. The improved properties of GQDs require the logical enactment of functional groups, which can be easily attained through post-synthetic non-covalent routes of modification. In this regard, the present work has for the first time employed a simple one-pot post-modification method utilizing the salt of amino caproic acid, an FDA approved reagent. The adsorption of the modifier on GQDs with varying weight ratios is characterized through DLS, zeta potential, Raman, absorption and fluorescence spectroscopy. A decrease of 20% in the fluorescence intensity with an increase in the modifier ratio from 1 to 1000 and an increased DLS size as well as zeta potential demonstrate the efficient modification as well as higher stability of the modified GQDs. The modified GQDs with a high weight ratio (1 : 100) of the modifier showed superior ability to sense dopamine, a neurotransmitter, as well as competent biofilm degradation ability. The modified GQDs could sense more efficiently than pristine GQDs, with a sensitivity as low as 0.06 μM (limit of detection) and 90% selectivity in the presence of other neurotransmitters. The linear relationship showed a decrease in the fluorescence intensity with increasing dopamine concentration from 0.0625 μM to 50 μM. Furthermore, the efficiency of the modified GQDs was also assessed in terms of their antibiofilm effect against

Seasonal and occupational exposure of the human body to extreme cold temperatures can result in cell death in the exposed area due to the formation of ice crystals. This leads to superficial or deep burn injury and compromised functionality. Currently available therapeutics can be ineffective in extreme cases, and thus, it is necessary to develop prophylactic strategies. In this study, we have devised a combination of known synthetic cryopreservative agents (termed SynAFP) and evaluated their potential antifreeze applications on skin. The prophylactic activity of SynAFP in vitro is indicated by improved cellular revival and cell viability, retention of the cytoskeleton, and normal cell cycle progression even after cold stress. A comprehensive whole-cell proteomic approach revealed that in the presence of SynAFP, cold-induced downregulation of proteins involved in cell-cell adhesion and upregulation of those related to mitochondrial stress were ameliorated. Pre-application of SynAFP in mice facing a frostbite challenge prevents their skin from incurring significant injury as confirmed through macroscopic and histological examination. Moreover, multiple applications of SynAFP on mouse skin at room temperature did not compromise skin integrity. SynAFP was also formulated in an

The world is experiencing one of the major viral outbreaks of this millennium, caused by a plus sense single-stranded RNA virus belonging to the Coronaviridae family, COVID-19, declared as pandemic by WHO. The clinical manifestations vary from asymptomatic to mild symptoms like fever, dry cough, and diarrhea, with further increase in severity leading to the development of acute respiratory distress syndrome. Though primary manifestations are respiratory and cardiac, various studies have shown the neuroinvasive capability of this virus resulting in neurological complications, which sometimes can precede common typical symptoms like fever and cough. Common neurological symptoms are headache, dizziness, anosmia, dysgeusia, confusion, and muscle weakening, progressing toward severe complications like cerebrovascular disease, seizures, or paralysis. Older adults and critically ill people are in the high risk group and have shown severe neurological symptoms upon infection. COVID-19 also has a profound impact on the mental health of people across the world. In this review, we briefly discuss the neurological pathologies and psychological impact due to COVID-19, which has not only stressed the physical health of people but has also created social and economic problems resulting in mental health issues.

Zinc oxide nanoparticles (ZnO NPs) are widely used in biomedicine and scientific research because of their high dissolution property and bioavailability. On the contrary, this property also increases the intracellular reactivity, accessibility and cytotoxicity. These nano-bio interactions could induce undesirable changes in the proteome of the interacting cells, especially in the lung cells as these are the primary contact site. However, the potential effects of ZnO NPs exposure on proteome remain unclear. Proteomics data will substantiate the detailed mechanism of cellular interactions and modulatory effects of ZnO NPs on cells. Quantitative proteomic profiling was done using MALDI-TOF/TOF and MS/MS to identify differential protein expression on exposure to NPs among non exposed and exposed cells. Twenty-two proteins, with approximately 1.5 fold differential expression in cells exposed to ZnO NPs as compared to control cells were identified. Differentially expressed proteins were further classified using PANTHER software on the basis of functional gene ontology term: molecular function, biological process and cellular component. ToppGene suite was used to study protein-protein interaction and network was enriched with STRING. This study is a systematic analysis of protein modulation of the A549 cells exposed to ZnO NPs indicating alterations in the cytoskeleton.

ZnO nanoparticles (ZnO NPs) are widely used engineered nanomaterials. Due to induced genotoxicity, increased oxidative stress, and teratogenicity, these NPs have been reported to be toxic. In the present study, we emphasise the role of vital proteins in regulating ZnO NP-induced abnormal phenotypes, particularly the deformed thorax and single wing in the Drosophila melanogaster progeny fed on 0.1-10 mM ZnO NPs. To understand how protein expression regulates this particular phenotype on ZnO NPs exposure, toxicoproteomics profile of control and abnormal phenotype flies was generated using LC/MS/MS. Gene ontology enrichment studies of proteomics data were carried out using CLUEGO and STRAP software. The bioinformatics tool STRING was used to generate a protein-protein interaction map of key proteins of enrichment analysis. Following ZnO NP exposure, the differential expression of key proteins of the Wnt pathway was prominent. Altered expression of various proteins of the Wnt pathway (CaMKII), cytoskeleton (Actin), and calponin resulted in developmental defects in drosophila progeny. In addition, immunohistology studies showed a significant deviation in the expression of wingless protein of ZnO NPs treated larvae in comparison to control. According to these findings, the interaction of the wnt pathway and cytoskeletal proteins with ZnO NPs caused developmental abnormalities in the subsequent generation of drosophila, highlighting the transgenerational toxic effects of these nanoparticles.

Two sugar PEG-based amphiphilic copolymers have been synthesized by Novozym®-435-catalyzed greener solvent free transesterification reaction of diastereomeric 2,6-anhydro-3,4,5-tri-O-benzyl-glucoheptitol and 2,6-anhydro-3,4,5-tri-O-benzyl-mannoheptitol with PEG-1000 diethyl ester in 80 and 76% yields, respectively. It has been observed that the configurational change at one of the carbons of diastereomeric 2,6-anhydro-heptitols has significant bearing on the degree of polymerization and characteristics of the synthesized polymers. Thus, the study of physico-chemical characteristics of synthesized anhydroglucoheptitol- and anhydromannoheptitol-PEG copolymers revealed that their aqueous solution forms spherical micelles of hydrodynamic diameters 11.2 and 6.4 nm, respectively. Micelles derived from both the amphiphilic copolymers have the potential to solubilize the hydrophobic dye, Nile red. Furthermore, a fluorescence microscopy study revealed that Nile red encapsulated micelles of synthesized amphiphilic copolymers uniformly internalized in A549 cells and thus can be used as a cargo carrier in biological systems. The viability of A549 cells was found to be very high even at a concentration of 500 μg mL−1 of copolymer indicating that they are cyto-compatible.

Aluminum oxide (Al2O3) nanoparticles (NPs) have a wide number of applications which cause intentional and unintentional exposure to humans, making it important to understand the nano-bio interactio...

The model biological organismDevelopmental stages ofThe crawling ability of larvae was significantly (ELF-EMF has maximum effects on acute exposure of third stage instar larvae and chronic exposure (egg to adult fly stage). These results suggest that electromagnetic radiations, though, have become indispensible part of our lives but they plausibly affect our health.

Hypobaric Hypoxia (HH) is known to cause oxidative stress in the brain that leads to spatial memory deficit and neurodegeneration . For decades therapeutic hypothermia is used to treat global and focal ischemia in preserving brain functions that proved to be beneficial in humans and rodents. Considering these previous reports, the present study was designed to establish the therapeutic potential of hypothermia preconditioning on HH induced spatial memory, biochemical and morphological changes in adult rats. Male Sprague Dawley rats were exposed to HH (7620 m, ~ 282 mmHg) for 1, 3 and 7 days with and without hypothermic preconditioning. Spatial learning memory was assessed by Morris water maze (MWM) test along with evaluation of hippocampal pyramidal neuron damage by histological study. Oxidative stress was measured by studying the levels of nitric oxide (NO), reactive oxygen species (ROS), lipid peroxidation (LPO), oxidized and reduced glutathione (GSSG and GSH). Results of MWM test indicated prolonged path length and latency to reach the platform in HH groups that regained to normal in cold pre-treated groups. A likely neurodegeneration was evident in HH groups that lessen in the cold pre-treated groups. Hypothermic preconditioning prevented spatial memory impairment and neurodegeneration in animals subjected to HH via decreasing the NO, ROS and LPO compared to control animals. The GSH level and GSH/GSSG ratio was found to be higher in preconditioned animals as compared to respective HH exposed animals, indicative of redox scavenging and restoration of hippocampal neuronal structure as well as spatial memory. Therefore, hypothermic preconditioning improves spatial memory deficit by reducing HH induced oxidative stress and hippocampal neurodegeneration, hence can be used as a multi-target prophylactic measure to combat HH induced neurodegeneration.

Zinc oxide nanoparticles (ZnO NPs) are commonly used nanomaterials (NMs) with versatile applications from high-end technologies to household products. This pervasive utilisation has brought human in the close interface with nanoparticles (NPs), hence questioning their safety prior to usage is a must. In this study, we have assessed the effects of chronic exposure to ZnO NPs (

Indocyanine green (ICG) is a near-infrared dye for wide-ranging applications, but its utility for biological studies is limited due to poor aqueous stability and concentration-dependent aggregation. Aqueous polymeric micelles/micellar aggregates of amphiphilic copolymers could be used to protect indocyanine green (ICG) by encapsulation, which is used in laser-mediated photothermal therapy (PTT) and photodynamic therapy (PDT). The sugar–PEG-based amphiphilic copolymers were synthesized using Novozym 435-catalyzed transesterification reaction under bulk conditions for encapsulation of ICG. A dye loading study revealed that micelles derived from copolymers having decanoylated and myristoylated sugar moieties encapsulate 62% and 92% of the ICG, respectively, with a multi-fold increase in its aqueous stability. Furthermore, an efficient internalization of micelles of acylated amphiphilic copolymers in aqueous medium was demonstrated by incubating cells with Nile red-encapsulated amphiphiles.

Systemic hypobaric hypoxia is reported to cause renal damage; nevertheless the exact pathophysiological mechanisms are not completely understood. Therefore, the present study aims to explore renal pathophysiology by using proteomics approach under hypobaric hypoxia. Six to eight week old male Sprague Dawley rats were exposed to hypobaric hypoxia equivalent to altitude of 7628 metres (pO2-282mmhg) at 28°C and 55% humidity in decompression chamber for different time intervals; 1, 3, and7 days. Various physiological, proteomic and bioinformatic studies were carried out to examine the effect of chronic hypobaric hypoxia on kidney. Our data demonstrated mild to moderate degenerative tubular changes, altered renal function, injury biomarkers and systolic blood pressure with increase in duration of hypobaric hypoxia exposure. Renal proteomic analysis showed 38 differential expressed spots, out of which 25 spots were down regulated and 13 were up regulated in 7 dayhypobarichypoxic exposure group of rats as compared to normoxia control. Identified proteins were involved in specific molecular changes pertinent to endogenous redox pathways, cellular integrity and energy metabolism. The study provides an empirical evidence of renal homeostasis under hypobaric hypoxia by investigating both physiological and proteomics changes. The identification of explicit key proteins provides a valuable clue about redox signalling mediated renal damage under hypobaric hypoxia.

Ensuring the nutritional security of combatants guarding the extreme altitudes along the Himalayan frontiers remains a daunting task owing to the harsh cold desert climatic conditions, landlocked terrain and sparse vegetation. These cold arid regions are characterized by rugged topography, extreme temperature variations, low precipitation, high wind velocity, thin atmosphere with high UV-radiation and fragile ecosystem. The Defence Life Science Journal decides to bring out a special issue on the theme ‘Cold Arid Agro-Animal Technologies’ focusing on the significance of agro-animal technologies for nutritional security of the soldiers deployed in inhospitable environments as well as the local population who have been living and thriving in these harsh conditions of high altitude.

Understanding the molecular basis of wound healing and tissue regeneration continues to remain as one of the major challenges in modern medicine. There is absolute necessity to unveil the rather elusive mechanism with a special emphasis on the approaches to accelerate wound healing. Low frequency low intensity Pulsed electromagnetic therapy is evidenced to have a significant impact on wound repair and regeneration. It provides a non-invasive reparative technique to treat an injury. In vitro studies reported a significant effect of electromagnetic field on neovascularisation and angiogenesis. There are also many pieces of evidence which support its efficiency in reducing the duration of wound healing and improving the tensile strength of scars. Here, we compared the traditional stigma associated with pulsed electromagnetic fields and weighed them with its potential therapeutic effect on wound healing. Furthermore, we emphasized the need for more focused research to determine the therapeutic strategies and optimised parameters of pulsed electromagnetic field that can assure efficient wound healing and regeneration.

Rapid development of nanotechnology has revolutionsed various areas of conventional food science and food industry. The novel properties of nanoparticles (NPs) have led to increasing application of nanotechnology in food industry. Nanofood market have a variety of products like the creamy ice-cream, drinks with no fat, enhanced flavour with nutrients and better textured, coloured and fresh looking food. Continuous monitoring for food spoilage or contamination is possible too. Nanotechnology has transformed the food industries which claim health benefits along with better taste. With the increasing use of NPs especially in food products, where humans are in close contact of the engineered nanomaterials (NMs), it is important to ensure safety before use. Bio-nano interactions often result in novel reaction and formation of products leading to toxicity. NPs mediated toxicity mainly includes inflammation, oxidative damage and genotoxicity. Prolong use of these particles can cause detrimental effects on health. Presently, due to lack of appropriate guidelines and regulations for food nanotechnology there are uncertainties regarding risk identification. Hence, it is essential to evaluate the consequences of this technology in terms of general public and occupational health risks associated with the manufacture, use and disposal of NMs, before instigating the same in day to day use.

Mutations in the chloride channel cystic fibrosis transmembrane regulator (CFTR) cause cystic fibrosis, a genetic disorder characterized by defects in CFTR biosynthesis, localization to the cell surface, or activation by regulatory factors. It was discovered recently that surface localization of CFTR is stabilized by an interaction between the CFTR N terminus and the multidomain cytoskeletal protein filamin. The details of the CFTR-filamin interaction, however, are unclear. Using x-ray crystallography, we show how the CFTR N terminus binds to immunoglobulin-like repeat 21 of filamin A (FlnA-Ig21). CFTR binds to beta-strands C and D of FlnA-Ig21 using backbone-backbone hydrogen bonds, a linchpin serine residue, and hydrophobic side-chain packing. We use NMR to determine that the CFTR N terminus also binds to several other immunoglobulin-like repeats from filamin A in vitro. Our structural data explain why the cystic fibrosis-causing S13F mutation disrupts CFTR-filamin interaction. We show that FlnA-Ig repeats transfected into cultured Calu-3 cells disrupt CFTR-filamin interaction and reduce surface levels of CFTR. Our findings suggest that filamin A stabilizes surface CFTR by anchoring it to the actin cytoskeleton through interactions with multiple filamin Ig repeats. Such an interaction mode may allow filamins to cluster multiple CFTR molecules and to promote colocalization of CFTR and other filamin-binding proteins in the apical plasma membrane of epithelial cells.

The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5′-triphosphates (NTPs) into cancer cells. Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the 3H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone. Nanogels with a particle size of 100–300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic®-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2–2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs. Nanogels composed of Pluronic® F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy.

Activation of cytotoxic nucleoside analogues in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogues may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5'-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, the 5'-triphosphate of an antiviral nucleoside analogue, 3'-azido-2',3'-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analogue 5'-triphosphate using a one-pot procedure. The corresponding 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating IC50 values 130-200 times lower than those values for AZT alone. The exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic P85. Cellular trafficking of nanogel particles, contrasted by polyethylenimine-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was observed following interactions of drug-loaded nanogels with cellular membranes. A drug release mechanism triggered by interaction of the drug-loaded nanogels with phospholipid bilayer is proposed. The results illustrate therapeutic potential of the phosphorylated nucleoside analogues formulated in nanosized cross-linked polymeric carriers for cancer chemotherapy.

The acetylation of proteins in biological systems is largely catalyzed by specific acetyl transferases utilizing acetyl CoA as the acetyl donor. The enzymatic acetylation of proteins independent of acetyl CoA was unknown until we discovered a unique membrane-bound enzyme in mammalian cells catalyzing the transfer of acetyl groups from polyphenolic peracetates (PAs) to certain enzyme proteins, resulting in the modulation of their catalytic activities. Since for the enzyme, acetyl derivatives of several classes of polyphenols such as coumarins, flavones, chromones, and xanthones were found to be acetyl donors, the enzyme was termed as acetoxy drug: protein transacetylase (TAase). TAase was found to be ubiquitously present in tissues of several animal species and a variety of animal cells. Liver microsomal cytochrome P-450 (CYP), NADPH-cytochrome c reductase and cytosolic glutathione S-transferase (GST) were found to be the targets for TAase-catalyzed acetylation by the model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC). Accordingly, the catalytic activities of CYP-linked, mixed function oxidases (MFOs) and GST were irreversibly inhibited while the reductase was remarkably activated. In this report, we have reviewed the details concerning purification and characterization of TAase and the protein acetylation by DAMC. Quantitative structure–activity relationship (QSAR) studies concerning the specificities of various PAs to liver microsomal TAase and TAase-related biological effects have also been reviewed.

The evidences for the possible enzymatic transfer of acetyl groups (catalyzed by a transacetylase localized in microsomes) from an acetylated compound (acetoxy-4-methylcoumarins) to enzyme proteins leading to profound modulation of their catalytic activities was cited in our earlier publications in this series. The investigations on the specificity for transacetylase (TA) with respect to the number and positions of acetoxy groups on the benzenoid ring of coumarin molecule revealed that acetoxy groups in proximity to the oxygen heteroatom (at C-7 and C-8 positions) demonstrate a high degree of specificity to TA. These studies were extended to the action of TA on acetates of other polyphenols, such as flavonoids and catechin with a view to establish the importance of pyran carbonyl group for the catalytic activity. The absolute requirement of the carbonyl group in the pyran ring of the substrate for TA to function was established by the observation that TA activity, was hardly discernible when catechin pentacetate and 7-acetoxy-3,4-dihydro-2,2-dimethylbenzopyran (both lacking pyran ring carbonyl group) were used as the substrates. Further, the TA activity with flavonoid acetates was remarkably lower than that with acetoxycoumarins, thus suggesting the specificity for pyran carbonyl group in proximity to the oxygen heteroatom. The biochemical properties of flavonoid acetates, such as irreversible activation of NADPH cytochrome C reductase and microsome-catalyzed aflatoxin B-1 binding to DNA in vitro were found to be in tune with their specificity to TA. (C) 2002 Elsevier Science Ltd. All rights reserved.

We have earlier established in tissues of several mammalian animal species the existence of a novel membrane bound enzyme termed 7,8-diacetoxy-4-methylcoumarin (DAMC): protein transacetylase (TAase) that possibly transfers acetyl groups from the model acetoxy drug (DAMC) to certain enzyme protein viz. glutathione S-transferase (GST), cytochrome P-450 and NADPH cytochrome C reductase leading to the drastic modulation of their catalytic activities. We have in this report extended the studies to human tissue and characterized TAase from placenta. For this purpose placental microsomes were preincubated with DAMC along with the receptor protein (cytosolic GST) followed by the addition of the substrates of GST in order to quantify the catalytic activity of GST, the extent of inhibition of GST served as a measure of TAase. Placental TAase was also found to irreversibly activate NADPH cytochrome C reductase by DAMC. Placental enzyme activated the reductase even at very low concentration of DAMC. Iodoacetamide nearly abolished the placental TAase suggesting the presence of active thiol group in the enzyme and the TAase demonstrated hyperbolic kinetics. Kinetic constants obtained by varying the concentrations of either of the substrates DAMC or cytosolic GST characterized TAase catalysed reaction as the bimolecular reaction. Further studies are in progress to delineate the physiological importance of TAase in placenta.

Therapies to prevent onset and progression of pulmonary arterial pressure are not very effective yet. This study was designed to investigate the effects of a novel dihydropyrimidinone, ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) on pathogenesis of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). For the same purpose, rats were injected intraperitoneally (i.p.) a single dose (60 mg/kg) of MCT which led to development of PAH in 21 days. MCT insult caused high mortality, pulmonary vascular and parenchymal remodelling. Since the course of PAH pathogenesis is characterised by an early onset and progression phases, H-DHPM was administered i.p. at 30 mg/kg dosage in MCT pre-injected animals either from day 0 through day 21 or day 14 though day 21 of MCT injection in two separate treatment groups. H-DHPM significantly improved survival, prevented remodelling of pulmonary vasculature and parenchyma and subsequently ameliorated PAH pathogenesis. Moreover, we observed significant decrease in right ventricle hypertrophy, measured by wet weight of right ventricle (RV) divided by wet weight of left ventricle plus septum (LV+S), in H-DHPM treated groups as compared to MCT injected animals. These findings suggest H-DHPM not only prevented development of PAH but also treated the PAH pathogenesis in progressive phase. In conclusion, our data determines H-DHPM, might be a future drug for the prevention of PAH.

The ability of various acetoxy derivatives of 4-methylcoumarins to inhibit the genotoxic changes due to aflatoxin B(1) (AFB(1)) is reported here. Several 4-methylcoumarins (test compounds), such as 7,8-diacetoxy-4-methylcoumarin (DAMC), monoacetoxy-4-methylcoumarin (MAC), 5-N-acetyl-6-acetoxy-4-methylcoumarin (NAMC) and 7,8-dihydroxy-4-methylcoumarin (DHMC) were separately administered intraperitoneally (i.p.) to male wistar rats followed by AFB(1) administration i.p. or intratracheally (i.t.) (2-8 mg/kg b.wt.) and another dose of the test compound. The animals were sacrificed 26h after AFB(1) administration. From animals receiving AFB(1) i.p., bone marrow (BM) cells were isolated and stained with Mayer's haematoxylin and eosin. Micronuclei (MN) in BM were scored by light microscopy. From animals receiving AFB(1) i.t., bronchoalveolar lavage (BAL) was obtained, lung cells (LG) were isolated and stained with fluorochrome 6-diamidino-2-phenylindole (DAPI) for the analysis of MN, apoptotic bodies (AP) and cell cycle variations. Rats were separately treated with the vehicle DMSO to serve as the proper control. AFB(1) caused significant dose-dependent induction of MN in BM as well as LG. AP were observed in LG of rats receiving AFB(1) and was found to correlate with MN induction. DAMC injection caused significant decrease in AP due to AFB(1) in LG and MN in both BM and LG. The effectiveness of MAC was approximately half that of DAMC, thereby indicating that number of acetoxy groups on the coumarin molecule determine the efficacy. The fact that NAMC had no effect either on MN or AP indicate that neither acetoxy group at C-6 nor the N-acetyl group at C-5 facilitate the transfer of acetyl group to P-450 required for inhibition of AFB(1)-epoxidation. DHMC, the deacetylated product of DAMC had no normalizing effect on the induction of MN and AP. These findings confirm our earlier hypothesis that DAMC-mediated acetylation of microsomal P-450 (catalysing epoxidation of AFB(1)) through the action of microsomal transacetylase is responsible for the protective action of DAMC. The relative number and position of acetoxy groups on the coumarin nucleus determine the specificity to the transacetylase necessary for the chemopreventive action.

Our earlier studies documented the ability of 7,8-diacetoxy-4-methylcoumarin (DAMC) to cause irreversible inhibition of cytochrome P-450 linked mixed function oxidases (MFO) mediated by membrane bound DAMC: protein transacetylase. Since P-450 catalyzed oxidation of benzene is crucial to its toxic effects, the action of DAMC and related analogues were considered promising in preventing the genotoxicity due to benzene. For this purpose rats were pretreated with various acetoxy-4-methylcoumarins (test compounds), which was followed by the administration of benzene either intratracheally (IT) or intraperitoneally (IP), and sacrificed 26 h after the injection of benzene. The incidence of micronuclei (MN) in bone marrow (BM) and lung (LG) were assessed by light and fluorescent microscopy, respectively. A dose-dependent induction of MN in BM and LG cells was observed in rats administered with benzene. A significant reduction in benzene-induced MN in BM and LG was observed as a result of DAMC administration to rats; a higher dose of DAMC resulted in greater inhibition of clastogenic action of benzene as revealed by MN incidence. 7,8-dihydroxy-4-methylcoumarin (DHMC), the deacetylated product of DAMC, demonstrated relatively lesser potency to inhibit the clastogenic action of benzene. This observation is consistent with the ability of DAMC to inhibit the formation of benzene oxide as well as to scavenge the oxygen radicals formed during the course of benzene metabolism. The fact that DHMC can only scavenge the oxygen radicals and is ineffective in inhibiting benzene oxidation in vivo explains the reduced capability of dihydroxy coumarin to prevent MN due to benzene. 7-Acetoxy-4-methylcoumarin (MAC) inhibits the MN due to benzene being roughly 50% of that produced by DAMC. DAMC is also effective in normalizing the cell cycle alterations produced by benzene in BM and LG. These observations further substantiate our hypothesis that the biological effects of acetoxy coumarins are mediated by the action of membrane bound transacetylase that catalyzes the acetylation of concerned proteins. Teratogenesis Carcinog. Mutagen. 21:181-187, 2001.

The enzymatic transfer of acetyl groups from acetylated xenobiotics to specific proteins is a relatively grey area in the evergreen field of biotransformation of foreign compounds. In this paper, we have documented evidence for the existence of a transacetylase in liver microsomes that catalyses the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to glutathione S-transferase (GST), either purified or present in cytosol leading to the irreversible inhibition of GST. A simple procedure is described for the assay of transacetylase by preincubation of DAMC with liver microsomes and pure GST/liver cytosol, followed by the addition of 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH) in order to quantify GST activity by the conventional procedure. The extent of inhibition of GST by DAMC under the conditions of the assay is indicative of DAMC:protein transacetylase activity. Following the assay procedure described here, the transacetylase was shown to exhibit hyperbolic kinetics. The bimolecular nature of the transacetylase reaction was apparent by the demonstration of K m and ν max values. 7,8-Dihydroxy-4-methylcoumarin (DHMC), one of the products of transacetylase reaction was identified and quantified using the partially purified enzyme. The fact that p -hydroxymercuribenzoate (PHMB) and iodoacetamide abolished irreversible inhibition of GST upon the action of transacetylase on DAMC strongly characterized transacetylase as a protein containing thiol group at the active site. In addition, the relative specificities of acetoxy 4-methylcoumarins to transacetylase have been demonstrated.

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization.

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca 2+ -binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca 2+ . Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase . Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.

The therapeutic efficiency of anticancer nucleoside analogues (NA) strongly depends on their intracellular accumulation and conversion into 5′-triphosphates. Because active NATP cannot be directly administrated due to instability, we present here a strategy of nanoencapsulation of these active drugs for efficient delivery to tumors. Stable lyophilized formulations of 5′-triphosphates of cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP) encapsulated in biodegradable PEG-cl-PEI or F127-cl-PEI nanogel networks (NGC and NGM, respectively) were prepared by a self-assembly procedure. Cellular penetration, in vitro cytotoxicity, and drug-induced cell cycle perturbations of these nanoformulations were analyzed in breast and colorectal cancer cell lines. Cellular accumulation and NATP release from nanogel was studied by confocal microscopy and direct high-performance liquid chromatography analysis of cellular lysates. Antiproliferative effect of dFdCTP nanoformulations was evaluated in human breast carcinoma MCF7 xenograft animal model. Nanoencapsulated araCTP, dFdCTP, and FdUTP showed similar to NA cytotoxicity and cell cycle perturbations. Nanogels without drugs showed very low cytotoxicity, although NGM was more toxic than NGC. Treatment by NATP nanoformulations induced fast increase of free intracellular drug concentration. In human breast carcinoma MCF7 xenograft animal model, i.v. dFdCTP-nanogel was equally effective in inhibiting tumor growth at four times lower administered drug dose compared with free gemcitabine. Active triphosphates of NA encapsulated in nanogels exhibit similar cytotoxicity and cell cycle perturbations in vitro and faster cell accumulation and equal tumor growth-inhibitory activity in vivo at much lower dose compared with parental drugs, illustrating their therapeutic potential for cancer chemotherapy. [Mol Cancer Ther 2008;7(10):3373–80]

Cochaperones are essential for Hsp70- and Hsc70-mediated folding of proteins and include nucleotide-exchange factors (NEFs) that assist protein folding by accelerating ADP-ATP exchange on Hsp70. The cochaperone Bag2 binds misfolded Hsp70 clients and also acts as an NEF, but the molecular basis for its function is unclear. We show that, rather than being a member of the Bag domain family, Bag2 contains a new type of Hsp70 NEF domain, which we call the 'brand new bag' (BNB) domain. Free and Hsc70-bound crystal structures of Bag2-BNB show its dimeric structure, in which a flanking linker helix and loop bind to Hsc70 to promote nucleotide exchange. NMR analysis demonstrates that the client binding sites and Hsc70-interaction sites of the Bag2-BNB overlap, and that Hsc70 can displace clients from Bag2-BNB, indicating a distinct mechanism for the regulation of Hsp70-mediated protein folding by Bag2.

Background Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains. Results The 2.9 Å crystal structure of the CHIP U-box domain complexed with UbcH5a shows that CHIP binds to UbcH5 and Ubc13 through similar specificity determinants, including a key S-P-A motif on the E2 enzymes. The determinants make different relative contributions to the overall interactions between CHIP and the two E2 enzymes. CHIP undergoes auto-ubiquitination by UbcH5 but not by Ubc13-Uev1a. Instead, CHIP drives the formation of unanchored polyubiquitin by Ubc13-Uev1a. CHIP also interacts productively with the class III E2 enzyme Ube2e2, in which the UbcH5- and Ubc13-binding specificity determinants are highly conserved. Conclusion The CHIP:UbcH5a structure emphasizes the importance of specificity determinants located on the long loops and central helix of the CHIP U-box, and on the N-terminal helix and loops L4 and L7 of its cognate E2 enzymes. The S-P-A motif and other specificity determinants define the set of cognate E2 enzymes for CHIP, which likely includes several Class III E2 enzymes. CHIP's interactions with UbcH5, Ube2e2 and Ubc13-Uev1a are consistent with the notion that Ubc13-Uev1a may work sequentially with other E2 enzymes to carry out K63-linked polyubiquitination of CHIP substrates.

Strong evidence supports the idea that specific metabolites of estrogens, mainly catechol estrogen-3,4-quinones, can react with DNA to become endogenous initiators of breast, prostate, and other human cancers. Oxidation of the catechol estrogen metabolites 4-hydroxyestradiol (4-OHE2) and 2-OHE2 leads to the quinones, estradiol-3,4-quinone (E2-3,4-Q) and estradiol-2,3-quinone (E2-2,3-Q), respectively. The reaction of E2-3,4-Q with DNA affords predominantly the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua, whereas the reaction of E2-2,3-Q with DNA yields the newly synthesized depurinating adduct 2-OHE2-6-N3Ade. The N3Ade adducts are lost from DNA by rapid depurination, while the N7Gua adduct is lost from DNA with a half-life of approximately 3 h at 37 degrees C. To compare the relative reactivity of E2-3,4-Q and E2-2,3-Q, the compounds were reacted individually with DNA for 0.5-20 h at 37 degrees C, as well as in mixtures (3:1, 1:1, 1:3, and 5:95) for 10 h at 37 degrees C. Depurinating and stable adducts were analyzed. In similar experiments, the relative reactivity of 4-OHE2 and 2-OHE2 with DNA was determined after activation by lactoperoxidase, tyrosinase, prostaglandin H synthase (PHS), or 3-methylcholanthrene-induced rat liver microsomes. Starting with the quinones, the levels of depurinating adducts formed from E2-3,4-Q were much higher than that of the depurinating adduct from E2-2,3-Q. Similar results were obtained with lactoperoxidase or tyrosinase-catalyzed oxidation of 4-OHE2 and 2-OHE2, whereas with activation by PHS or microsomes, a relatively higher amount of the depurinating adduct from E2-2,3-Q was detected. These results demonstrate that the E2-3,4-Q is much more reactive with DNA than E2-2,3-Q. The relative reactivities of E2-3,4-Q and E2-2,3-Q to form depurinating adducts correlate with the carcinogenicity, mutagenicity, and cell-transforming activity of their precursors, the catechol estrogens 4-OHE2 and 2-OHE2. This is essential information for understanding the cancer risk posed by oxidation of the two catechol estrogens.

An enhanced intracellular level of Nitric oxide (NO) is essential to ameliorate several pathological conditions of heart and vasculature necessitating the activation of NOS. We have projected in this report the acetylation of eNOS by polyphenolic peracetates (PA) catalyzed by the novel enzyme acetoxy drug: protein transacetylase (TAase) discovered in our laboratory as an unambiguous way of activating NOS which results in the manifestation of physiological action. The human platelet was chosen as the experimental system in order to validate the aforementioned proposition. PA caused profound irreversible activation of platelet NADPH cytochrome c reductase mediated by TAase. The convincing biochemical evidences are presented to show that PA could cause acetylation of the reductase domain of NOS leading to the activation of eNOS in tune with their specificities to platelet TAase. As a result, the enhanced level of NO due to activation of platelet eNOS by PA was found to inhibit the ADP-induced platelet aggregation. The present studies highlight for the first time the role of PA as the novel potent agent for enhancing the intracellular NO levels.

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4- methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K m (1667 μM) and Vmax (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys51, Lys82, Lys 123, Lsy181, Lys191 and Lys210) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions. © 2003 Elsevier B.V. All rights reserved.

The quantitative structure-activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase) activity. Hence, an attempt was made to examine whether such substrate analogues of benzopyran acetates which lack in the pyran ring carbonyl group, such as 7-acetoxy-2,3-dihydro-2,2-dimethylbenzopyran (BPA), cetachin pentaacetate (CPA) and hematoxylin pentaacetate (HPA) could inhibit the 7,8-diacetoxy-4-methylcoumarin (DAMC):protein (glutathione-S-transferase) transacetylase activity. These compounds were indeed found to remarkably inhibit the TAase activity in a concentration dependent manner and exerted their inhibitory action very rapidly. Further BPA, CPA and HPA were found to abolish the TAase mediated activation of NADPH cytochrome C reductase as well as the inhibition of liver microsome catalyzed aflatoxin B(1) (AFB(1))-DNA binding by DAMC very effectively. These results strongly suggest that the acetoxybenzopyrans merit as potent inhibitors of TAase.

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B(1) (AFB(1)). We have in this report extended the observations to quercetin pentaacetate (QPA), which unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB(1) epoxidation as measured by AFB(1) binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB(1).

Our earlier work established a convenient assay procedure for acetoxycoumarin (AC): protein transacetylase (TA) by indirectly quantifying the activity of glutathione (GSH)-S-transferase (GST), the extent of inhibition of GST under the conditions of the assay represented TA activity. In this communication, we have probed the specificity for TA with respect to the number and position of acetoxy groups on the benzenoid as well as the pyranone rings of the coumarin system governing the efficient transfer of acetyl groups to the protein(s). For this purpose, coumarins bearing one acetoxy group, separately at C-3 or C-4 position and 4-methylcoumarins bearing single acetoxy group, separately at C-5, C-6 or C-7 position were synthesized and specificities to rat liver microsomal TA were examined. Negligible TA activity was discernible with 3-AC as the substrate, while the substrate efficiency of other AC were in the order 7-acetoxy-4-methylcoumarin (7 AMC)>6 AMC>5 AMC=5 ADMC=4 AC. To achieve a comparable level of GST inhibition which was proportional to the enzymatic transfer of acetyl groups to the protein (GST), the concentrations of 7-AMC, 6-AMC, 5-AMC and 4-AC were in the order 1:2:4:4, respectively. One diacetoxycoumarin, i.e., 7,8-diacetoxy-4-methylcoumarin (DAMC) was also examined and it was found to elicit maximum level of GST inhibition, nearly twice that observed with 7-AMC. These observations lead to the logical conclusion that a high degree of acetyl group transfer capability is conferred when the acetoxy group on the benzenoid ring of the coumarin system is in closer proximity to the oxygen heteroatom, i.e., when the acetoxy groups are at the C-7 and C-8 positions.

The current knowledge on biological protein acetylation is confined to acetyl CoA-dependent acetylation of protein catalyzed by specific acetyl transferases and the non-enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane-bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8-diacetoxy-4-methyl coumarin (DAMC) to glutathione S-transferase 3-3 (GST3-3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3-3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N-terminus and six lysines, Lys-51, -82, -124, -181, -191 and -210, were found to be acetylated. The acetylation of GST3-3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved. Chemicals/CAS: 7,8-diacetoxy-4-methylcoumarin; Acetyl Coenzyme A, 72-89-9; Acetylesterase, EC 3.1.1.6; Coumarins; Glutathione Transferase, EC 2.5.1.18; Recombinant Proteins

The potential role of polyphenolic acetate (PA) in causing diverse biological and pharmacological actions has been well studied in our laboratory. Our investigations, for the first time, established the role of calreticulin transacetylase (CRTAase) in catalyzing the acetylation of nitric oxide synthase (NOS) by Pas leading to robust activation of NOS. 7, 8- Diacetoxy-4-methylcoumarin (DAMC) and other acetoxycoumarins augmented the expression of thioredoxin (TRX) and vascular endothelial growth factor (VEGF) in human peripheral blood mononuclear cells (PBMCs). These findings substantiated our earlier observations that DAMC was a superb inducer of angiogenesis. The enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to increased expression and activity of TRX in PBMCs due to the action of DAMC was revealed by real time RT-PCR analysis. The possible activation of TRX due to acetylation was confirmed by the fact that TRX activity of PBMCs was enhanced by various acetoxycoumarins in tune with their affinities to CRTAase as substrates. DAMC caused enhanced production of NO by way of acetylation of NOS as mentioned above and thereby acted as an inducer of VEGF. Real time RT-PCR and VEGF ELISA results also revealed the overexpression of TRX. DAMC and other PAs were found to reduce the oxidative stress in cells as proved by significant reduction of intracellular ROS levels. Thus, the crucial role of TRX in DAMC-induced angiogenesis with the involvement of VEGF was established.