Kynurenines Dynamics in the Periphery and Central Nervous System Steers Behavioral Deficits in Rats under Hypobaric Hypoxia.

People travel to high-altitude regions as tourists, workers, and military personnel on duty. Despite the consistent 21% oxygen content in the atmosphere, ascending to higher altitudes results in a decrease in the partial pressure of oxygen, inducing a state known as hypobaric hypoxia (HH). HH is an environmental stress that is responsible for neuroinflammation and behavioral deficits (anxiety, depression, mood disturbance, etc.), but little is known about its metabolic pathways. The kynurenine pathway (KP) is a promising candidate to uncover the mysteries of HH stress, as it is an important regulator of the immune system and is associated with behavioral deficits. To investigate the role of KP under HH, the levels of KP metabolites in the serum, cerebrospinal fluid (CSF), and brain tissue (prefrontal cortex-PFC, neocortex, and hippocampus) of male Sprague-Dawley rats exposed to HH at 7620 m for 1, 3, and 7 days were estimated utilizing high-performance liquid chromatography (HPLC). The behavioral analogs for anxiety-like and depression-like behavior were assessed using the open field test and forced swim test, respectively. Upon HH exposure, crosstalk between the periphery and central nervous system and KP metabolite region-dependent differential expression in the brain were observed. KP metabolites showed a positive correlation with behavioral parameters. The results of our study are indicative that KP can be proposed as the etiology of behavioral deficits, and KP metabolite levels in serum or CSF can be used as plausible markers for anxiety-like and depression-like behaviors under HH stress with a scope of targeted therapeutic interventions.

Anti-microbial efficacy of a scientifically developed and standardized herbal-alcohol sanitizer.

Hands are the primary mode of transmission of microbe-based infections, as they harbor normal microbiota and pathogenic microbes. SARS-CoV-2 has endangered lives worldwide, and WHO has recommended good hygiene practices, especially hand hygiene. In addition, other infectious diseases like diphtheria, measles, tuberculosis, HIV, malaria, etc. are spreading in the shadow of the COVID-19 pandemic. The anti-microbial efficiency of two in-house developed herbal-alcohol based hand sanitizers containing Azadirachta indica, Citrus limon, Zingiber officinale, and Aloe vera (HS1) and Zingiber officinale replaced with Ocimum sanctum (HS2) was evaluated. HS1, with Zingiber officinale, and HS2, with Ocimum sanctum, herbal sanitizers showcased in-vitro anti-viral activity on MDCK cells using the reference strain of influenza A virus, A/PR/8/34 (H1N1), and reduced 99.99% of microbial load within 30 s of contact time, estimated by the Antimicrobial Susceptibility Testing Method. On volunteers, HS1 and HS2 were more effective than alcohol-based WHO sanitizers. Moreover, HS2 sanitizer is more effective against viruses and has better efficiency and hedonic qualities in volunteers than HS1. These sanitizers don't irritate or dry up the skin and have a longer shelf life. Overall, findings reveal that herbal-alcohol-based sanitizers are promising hand hygiene products with the capability of reducing microbial load.

Zinc oxide nanoparticles trigger dysfunction of mitochondrial respiratory complexes and repair dynamics in human alveolar cells.

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 interplay of calponin, wnt signaling, and cytoskeleton protein governs transgenerational phenotypic abnormalities in drosophila exposed to zinc oxide nanoparticles.

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.

Post-synthetic modification of graphene quantum dots bestows enhanced biosensing and antibiofilm ability: efficiency facet.

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 Staphylococcus aureus. The unmodified GQDs showed only 10% disruption of the adhered bacterial colonies, while the modified GQDs (1 : 100) showed significantly more than 60% disruption of the biofilm, presenting the competency of the modified GQDs. The unique modifications of GQDs have thus proven to be an effective method for the proficient utilization of zero-dimensional carbon nanomaterials for biosensing, bioimaging, antibacterial and anti-biofilm applications.

Alteration in cerebral blood flow, kynurenines with respect to mood profile in freshly recruited armed forces personnel.

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.

A Combination of Synthetic Molecules Acts as Antifreeze for the Protection of Skin against Cold-Induced Injuries.

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 anAloe vera-based cream (referred to as fSynAFP), which offered similar protection under cold stress conditions. Thus, SynAFP can be considered as a potential candidate for formulating a topical intervention for protection from cold-induced injuries to skin.

Hypothermic preconditioning attenuates hypobaric hypoxia induced spatial memory impairment in rats.

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.

Effects of extremely low-frequency electromagnetic field on different developmental stages of Drosophila melanogaster.

PURPOSE: The model biological organism Drosophila melanogaster has been utilized to assess the effect of extremely low-frequency electromagnetic field (ELF-EMF) on locomotion, longevity, developmental dynamics, cell viability and oxidative stress. MATERIALS AND METHOD: Developmental stages of Drosophila melanogaster (Oregon R strain) individually exposed to ELF-EMF (75 Hz, 550 µT) for 6 h once for acute exposure. For chronic exposure, complete life cycle of fly, that is, egg to adult fly was exposed to ELF-EMF for 6 h daily. The effect of exposure on their crawling and climbing ability, longevity, development dynamics, cellular damage and oxidative stress (generation of reactive oxygen species (ROS)) was evaluated. RESULTS: The crawling ability of larvae was significantly (p < .05) reduced on acute (third stage instar larvae) as well as chronic exposure (F0 and F1 larvae). When locomotion of flies was tested using climbing assay, no alteration was observed in their climbing ability under both acute and chronic exposure; however, when their speed of climbing was compared, a significant decrease in speed of F1 flies was observed (p = .0027) on chronic exposure. The survivability of flies was significantly affected under chronic and acute exposure (at third stage instar larvae). In case of acute exposure of the third stage instar larvae, although all the flies were eclosed by the 17th day, there was a significant decline in the number of flies (p = .007) in comparison to control. While in case of chronic exposure apart from low number of flies eclosed in comparison to control, there was delay in eclosion by one day (p = .0004). Using trypan blue assay, the internal gut damage of third stage instar larvae was observed. Under acute exposure condition at third stage instar larvae, 30% larvae has taken up trypan blue, while only 10% larvae from acute exposure at adult stage. On chronic exposure, 50% larvae of the F1 generation have taken up trypan blue. On evaluation of oxidative stress, there is a significant rise in ROS in case of acute exposure at third stage instar larvae (p = .0004), adult fly stage (p = .0004) and chronic exposure (p = .0001). CONCLUSION: 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.

Brain and COVID-19 Crosstalk: Pathophysiological and Psychological Manifestations.

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.

Aluminum oxide nanoparticles mediated toxicity, loss of appendages in progeny of Drosophila melanogaster on chronic exposure.

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 interaction. In this study, we made an attempt to evaluate the toxic effects of Al2O3 NPs chronic exposure on Drosophila melanogaster. Flies were exposed to Al2O3 NPs at concentration 0.1 and 1 mM via ingestion throughout their lifespan and progeny flies were screened for behavioral and phenotypic abnormalities. Behavioral abnormalities in flies were recorded through larval crawling, climbing in flies and two taste testing. Chronic exposure of Al2O3 NPs resulted in the loss of appendages in flies resulting in five legs flies, four legs flies and absence of haltere. Exposure to Al2O3 NPs caused renal failure in flies as observed by swollen abdomen. Our observations clearly showed that these NPs could cause detrimental health ailments which relate to human birth deformities and kidney failure. Damage at the cellular level was studied through proteomic profiling. Three hundred and seven unique proteins were expressed on exposure to Al2O3 NPs and 51 proteins were differentially expressed. Enrichment analysis of differentially expressed proteins showed significant alteration in striated muscle cell differentiation, digestive tract morphogenesis, phototransduction, regulation of chromatin organization and DNA duplex unwinding.

Hypobaric hypoxia induced renal damage is mediated by altering redox pathway.

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.

Corrigendum to The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

In the Original Research Article entitled "The Competence of 7, 8-Diacetoxy-4-methylcoumarin and other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis" Published in Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 2, on page no. 179, the order of author names was rearranged because second authorship is acceptable as they only acknowledge the first and the second authorship as per the new policies of Medical Council of India. The order of authors should be read as follows: Rini Joshi, Vishwajeet Rohil, Shvetambri Arora, Sushma Manral, Ajit Kumar, Sanjay Goel, Nivedita Priya, Prabhjoth Singh, Prija Ponnan, Suvro Chatterji, Bilikere S. Dwarakanath, Daman Saluja, Diwan S. Rawat, Ashok K. Prasad, Luciano Saso, Ekta Kohli, Anthony L. DePass, Marc E. Bracke, Virinder S. Parmar and Hanumantharao G. Raj.

Chronic exposure of zinc oxide nanoparticles causes deviant phenotype in Drosophila melanogaster.

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 (<50nm) on the model organism Drosophila melanogaster. Potential toxic effects were studied by evaluating longevity, climbing ability, oxidative stress and DNA fragmentation. Ensuing exposure, the F0 (parent), F1, F2, F3 and F4 generation flies were screened for the aberrant phenotype. Flies exposed to ZnO NPs showed distinctive phenotypic changes, like deformed segmented thorax and single or deformed wing, which were transmitted to the offspring's in subsequent generations. The unique abnormal phenotype is evident of chronic toxicity induced by ZnO NPs, although appalling, it strongly emphasize the importance to understand NPs toxicity for safer use.

Hypobaric Hypoxia Imbalances Mitochondrial Dynamics in Rat Brain Hippocampus.

Brain is predominantly susceptible to oxidative stress and mitochondrial dysfunction during hypobaric hypoxia, and therefore undergoes neurodegeneration due to energy crisis. Evidences illustrate a high degree of association for mitochondrial fusion/fission imbalance and mitochondrial dysfunction. Mitochondrial fusion/fission is a recently reported dynamic mechanism which frequently occurs among cellular mitochondrial network. Hence, the study investigated the temporal alteration and involvement of abnormal mitochondrial dynamics (fusion/fission) along with disturbed mitochondrial functionality during chronic exposure to hypobaric hypoxia (HH). The Sprague-Dawley rats were exposed to simulated high altitude equivalent to 25000 ft for 3, 7, 14, 21, and 28 days. Mitochondrial morphology, distribution within neurons, enzyme activity of respiratory complexes, Δψ m , ADP: ATP, and expression of fission/fusion key proteins were determined. Results demonstrated HH induced alteration in mitochondrial morphology by damaged, small mitochondria observed in neurons with disturbance of mitochondrial functionality and reduced mitochondrial density in neuronal processes manifested by excessive mitochondrial fragmentation (fission) and decreased mitochondrial fusion as compared to unexposed rat brain hippocampus. The study suggested that imbalance in mitochondrial dynamics is one of the noteworthy mechanisms occurring in hippocampal neurons during HH insult.

The Competence of 7,8-Diacetoxy-4-Methylcoumarin and Other Polyphenolic Acetates in Mitigating the Oxidative Stress and their Role in Angiogenesis.

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.

The Competence Of 7,8-Diacetoxy-4-Methylcoumarinand Other Polyphenolic Acetates In Mitigating The Oxidative Stress And Their Role In Angiogenesis.

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 variousacetoxycoumarins 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.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator.

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.

Biochemical basis of the interaction between cystic fibrosis transmembrane conductance regulator and immunoglobulin-like repeats of filamin.

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.

Calreticulin transacylase: genesis, mechanism of action and biological applications.

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 55kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca2+-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 Ca2+. 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.