Reduction In Specific Activity Research Articles

Enzyme inhibitory potency of nano formulation of some plant oils on the red palm weevil Rhynchophorus ferrugineus olivier

Inhibitory effect of the tested plant oils and their nanoformulation against trypsin and chymotrypsin enzyme extracted from Rhynchophorus ferrugineus, was studies in in vivo and in vitro. In vivo inhibition, the reduction in the trypsin specific activity caused by the bulk oils was greater than that of chymotrypsin enzyme. Compared to control nanoformulation caused significant reduction in the specific activity of trypsin enzyme; however, there were no inhibition effect observed on chymotrypsin enzyme. In vitro bulk oils showed very low inhibitory effect against trypsin enzyme, while they showed strong inhibition activity against chymotrypsin with recording for ginger nanoformulation (half-maximal inhibitory concentration (IC50) =6.83 μg/ml), purple nutsedge (7.632 μg/ml), garden cress (11.52 μg/ml) and castor (16.82 μg/ml), nanoformulation of these oils showed uncompetitive type of inhibition as the Vmax and Km values changed compared to control. In conclusion, nanoformulation of the tested plant oils could be used in the integrated pest management (IPM) programs of the red palm weevil (RPW).

Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa

Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and kcat/Km values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the KmGSH and I50 values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (kcat/KmCDNB) plot was used to estimate the pKa value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pKa value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pKa of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.

The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase

All malate dehydrogenases have a flexible loop that starts to close over the active site region of malate dehydrogenase when cofactor binds and locks down completing the active site when the dicarboxylic acid substrate binds. As such the active site loop appears to play a role in both substrate specificity and correct alignment of the substrate to allow hydride transfer and proton abstraction/donation depending on the direction of the reaction. Combined with available crystal structures, detailed analysis of the 1smk.pdb structure shows the loop in a series of microstates from fully open to a closed state induced by the inhibitor citrate binding (figure 1), sequence conservation suggests that the loop region contains a 19 amino acid stretch from P119 to N137 using the watermelon glyoxysomal enzyme as reference. Clustal Omega Analysis of malate dehydrogenases from prokaryotes to mammals shows 5 families of conservation of the loop region (figure 2) allowing a series of hypotheses about structure function relationships of specific residues within the sequence to be made which have been tested by a combination of computational and site directed mutagenesis wetlab approaches. In addition to the two canonical arginines involved in substrate binding, we have explored every other residue in the loop region. Overall, mutations in the first half of the loop had small impact on Specific Activity while mutations in the second half of the loop had larger impacts. R130 mutants to E or S had the most impact while mutation to A or Q had lesser effects. Three other residues in particular had striking effects on Oxaloacetate saturation, G121A, L133S and N137. L133 and N137, at the C terminal base of the loop, were hypothesized to play a role in the repulsive interactions between substrate and cofactor in the active site. Consistent with this hypothesis, creation of an L133S or D137A mutant showed that concomitant with a reduction in specific activity to 2% and 5% wildtype (respectively), Km for Oxaloacetate was dramatically increased while Km for NADH was decreased somewhat. Finally, a series of mutations of M128 (Q,I and A) were explored with varying effects on specific activity (Q>I>A) and significant effects on Km for Oxaloacetate but lesser or no effects on Km for NADH. The impact of M128 mutations in particular were further explored computationally using DeepDDG analysis and effects on stability correlated with impact on kinetic properties. Overall these studies provide a detailed picture of the complex interactions the flexible loop makes that contribute to both substrate binding and catalysis.

Inhibition of the metabolism of mixed cultures of purple phototrophic bacteria by typical refinery and petrochemistry wastewater pollutants

AbstractBACKGROUNDAnaerobic treatments based on purple phototrophic bacteria (PPB) can be a more efficient and less energetically demanding alternative to aerobic biological processes currently used in refinery and petrochemistry wastewater (RPWW) treatment plants. Toxic compounds in the RPWW might compromise PPB metabolism, limiting its feasibility. In this work, seven toxic compounds that are typical components of RPWW [benzene, toluene, ethylbenzene, o‐xylene (BTEX), furfural, methyl‐di‐ethanolamine (MDEA) and butanone] were evaluated as potential inhibitors of the specific activity and growth of PPB.RESULTSInhibitory Ki parameters of 50% reduction of specific activity or biomass yield were calculated. Most of the BTEX compounds showed initial inhibitory effects on the specific activity, with Ki values of 305 ± 9, 151 ± 17 and 84 ± 2 mg L−1 for benzene, toluene and ethylbenzene, respectively. These compounds also caused inhibition of PPB growth (Ki of 411 ± 7 and 241 ± 3 mg L−1, 26% below the control, respectively). By contrast, o‐xylene affected neither the activity nor the growth. Furfural inhibited the initial specific activity (Ki of 448 ± 24 mg L−1), with no effect over the PPB growth. However, MDEA and butanone improved the initial specific activity of PPB (15% and 40% above the control, respectively). However, butanone negatively impacted the growth which decreased to ≤40% below the control.CONCLUSIONSCalculated inhibition thresholds for the seven toxic compounds were far above the actual concentrations found in the RPWW. Thus, anaerobic treatments based on PPB for the treatment of RPWW would not be limited by the presence of the toxic compounds studied here. © 2021 Society of Chemical Industry (SCI).

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

Зміна вмісту 137CS у корі крушини ламкої (Frangula Alnus Mill.) у вологих суборах лісів Полісся України з часу аварії на ЧАЕС

Досліджено динаміку вмісту 137Cs у корі крушини ламкої у лісах Житомирського Полісся з часу аварії на ЧАЕС (1991-2018 рр.). Відзначено істотне зменшення величини питомої активності 137Cs у корі крушини ламкої на постійних пробних площах за період спостережень (27 років) – від 4 до 38 разів, тобто темпи зниження питомої активності 137Cs у корі крушини ламкої на різних постійних пробних площах відрізняються у широких межах. Це можна пояснити особливостями екологічних умов кожної пробної площі, а також недосконалістю методичних підходів до відбору зразків. Зменшення питомої активності радіонукліда в корі крушини ламкої зумовлено: природним розпадом 137Cs у ґрунті та в самій корі; жорсткішим закріпленням радіоактивного елементу у ґрунті; зменшенням активності радіонукліда у ґрунті завдяки його надходженню до чисельних компонентів лісових біогеоценозів. Також відзначено зниження рівня щільності радіоактивного забруднення ґрунту – від 2,2 до 3,8 раза, що пояснено розпадом 137Cs, його жорсткішим закріпленням у ґрунті та переміщенням його частини до лісових рослин. Зазначено особливості методичних підходів до відбору зразків ґрунту для визначення величини щільності радіоактивного забруднення ґрунту, – враховуючи вертикальну міграцію 137Cs у нижчі шари ґрунту, тепер зразки ґрунту необхідно відбирати завглибшки 25 см (на відміну від відбору зразків ґрунту завглибшки 10 см). Виявлено сучасні рівні радіоактивного забруднення та встановлено залежність між щільністю радіоактивного забруднення ґрунту та вмістом радіонукліда а корі крушини ламкої. Простежено лінійну залежність збільшення концентрації 137Cs у корі крушини із збільшенням величини щільності радіоактивного забруднення ґрунту. Крушину ламку віднесено до рослин, які помірно накопичують 137Cs.

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility.

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in KM and kcat The catalytic efficiency (kcat/KM) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.

Mapping the O‐GlcNAc‐Cytoprotective network

In the 30 years since the discovery of nucleocytoplasmic glycosylation, O‐GlcNAc has been demonstrated to regulate protein function by modulating characteristics that include protein folding, localization, degradation, activity, post‐translational modifications, and interactions. The cell coordinates these molecular events, on thousands of cellular proteins, in concert with environmental and physiological cues to fine‐tune epigenetics, transcription, translation, signal transduction, the cell cycle, and metabolism. The cellular stress response is no exception: diverse forms of injury result in dynamic changes to the O‐GlcNAc sub‐proteome that promote survival. To date, the majority of studies have focused on identifying proteins and pathways regulated by O‐GlcNAc that combat cytotoxicity. Currently, little is known about the regulation of the two enzymes that write and erase the O‐GlcNAc‐modification, the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA). To provide insight into the regulation of OGA, the enzyme that catalyzes the removal of O‐GlcNAc, proximity biotin ligation (BioID) in combination with Stable Isoptope Labeling of Amino Acids in Cell Culture (SILAC) was used to define the basal and stress‐induced interactome of OGA. This analysis revealed 90 interaction partners of OGA, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase, OGT, heat shock cognate 70 kDa protein (HSC70), filamin‐A, and heat shock protein 27 (HSP27) were confirmed by co‐immuno‐precipitation. Furthermore, the pool of OGA bound to FAS demonstrated a substantial reduction in specific activity (~85%), suggesting that FAS is a novel protein inhibitor of OGA. Consistent with these observations, overexpression of FAS augmented O‐GlcNAcylation of a subset of cellular proteins. Collectively, these data support a model in which protein interactors of OGA, such as FAS, regulate its local activity and thus lead to remodeling of the O‐GlcNAc‐sub proteome in a manner consistent with cell survival.Support or Funding InformationThis work was supported by grants from the NIH National Heart, Lung, and Blood Institute (P01HL107153), the NIH National Institute on Aging (F31AG047724), and the NIH National Institute of General Medical Sciences (MARC U‐STAR; T34GM008663). Core Facility support was provided NIH Grant S10 RR024550 ‐ Research Resources (JHUSOM Microscope Facility) and NIH Grant 2P30 CA006973 ‐ National Cancer Institute (JHUSOM Mass Spectrometry and Proteomics Facility).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of perchloric acid as an oxidative stress trigger for protein threonine phosphatase in Arachis hypogaea L.

The protein threonine phosphatases (PThPases) catalyze the reversible phosphorylation of threonine residues in proteins. They also regulate a large number of cellular processes including developmental and signaling pathways in eukaryotes.This study was conducted to determine the activity of PThPases as affected by different oxidizing agents in peanut ( Arachis hypogaea L.) seedlings. In present work, the peanut seedlings were grown in dark on moistened whatman filter paper at 28±2°C and it was observed that the activity of PThPase increased several folds in 6-8 days growing seedlings (without stressed). The eight days growing seedlings were then further treated with 2 per cent solution of different oxidizing agents (Hydrogen peroxide, Nitric acid, Potassium dichromate, Perchloric acid, Potassium permagnate, Magnese dioxide, Lead dioxide and Ammonium persulphate).It was observed that the specific activity of PThPase got an appreciable reduction by perchloric acid (HClO 4 ). After treating the 8 days growing seedlings with different % HClO 4 (0.5-5%), it was observed that 3% HClO 4 shows a pronounced effect on PThPase activity and the specific activity was reduced upto 65 per cent. In addition,when seedlings were treated with 3% HClO 4 for different time intervals (1-6h),4h treatment causes more than 88 per cent reduction in specific activity. The different parts of seedling (stressed with 3% HClO 4 for 4h) show the maximum reduction in specific activity in hypocotyl followed by epicotyl, cotyledon and then root. The results suggest that the role of PThPase is stress-related cellular processes and likely to inhibit the activity of intracellular protein threonine phosphatases.

Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress

The dynamic post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc, respectively, are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination with stable isotopic labeling of amino acids in cell culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70-kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation.

Abstract 575: Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types

Abstract The cytokine TWEAK, a TNF superfamily member, and its cognate receptor, Fn14, have emerged as potentially valuable targets for cancer therapy since their expression has been frequently identified as independent negative prognostic indicators in a variety of tumor types. The serine protease Granzyme B (GrB) is a highly cytotoxic component of human immune effector cells and induces multiple, intense pro-apoptotic signals when delivered to the cytoplasm of target cells. This human protein has been well-studied, operating through multimodal pathways which are both caspase-dependent and caspase-independent. Our laboratory has designed series of new Fn14 targeted fusion constructs containing an engineered GrB payload. The GrB-Fc-IT4 containing either WTCYS or EACYS mutants of GrB, and a human scFv targeting Fn14 were expressed in HEK293E and suspension CHO cells, harvested under serum-free conditions and purified to homogeneity. Both constructs display high-level protein production (>50 mg/L), enhanced serum stability and impressive cytotoxic activity (IC50 < 20 nM) against Fn14 positive cells. The specific activity of the GrB moiety, as assessed by cleavage of a synthetic chromogenic GrB substrate, was comparable to that of human GrB for the WTCYS while the EACYS variant showed a 50% reduction in specific activity. On the other hand, in vitro stability studies showed that EACYS was more stable after 48h incubation in human serum, retaining over 60% of its initial specific activity. Against a panel of 20 human cell lines expressing Fn14, both constructs showed high affinity and selective cytotoxicity within the nanomolar range (IC50 ranged from 4 to 284 nM) and were two to over a hundred times more potent than free GrB. Mechanistic studies demonstrated that GrB-Fc-IT4 constructs activated caspase cascades and cytochrome C related pro-apoptotic mechanisms in keeping with the known intracellular functions of GrB in target cells. Pharmacokinetic studies in mice revealed that GrB-Fc-IT4 fusion proteins exhibited a bi-exponential clearance from plasma with a rapid initial clearance (t ½ α = 0.36 hours) followed by a prolonged terminal-phase plasma half-life (t 1/2 β = 35 hours). Toxicity studies in mice demonstrated that the MTD is above 100 mg/kg total dose. Murine orthotropic tumor model efficacy and bio-distribution studies are ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Ana Alvarez-Cienfuegos, Lawrence H. Cheung, Khalid A. Mohamedali, Yunli Zhao, Kathryn E. Ruisaard, Jeffrey A. Winkles, Michael G. Rosenblum. Human serine protease therapeutics- potent constructs targeting Fn14 in multiple tumor types. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 575.

Elucidation of structural and functional properties of albumin bound to gold nanoparticles

Nanoparticle–albumin complexes are being designed for targeted drug delivery and imaging. However, the changes in the functional properties of albumin due to adsorption on nanoparticles remain elusive. Thus, the objective of this work was to elucidate the structural and functional properties of human and bovine serum albumin bound to negatively charged gold nanoparticles (GNPs). Fluorescence data demonstrated static quenching of albumin by GNP with the quenching of buried as well as surface tryptophan in BSA. The binding process was enthalpy and entropy-driven in HSA and BSA, respectively. At lower concentrations of GNP there was a higher affinity for tryptophan, whereas at higher concentrations both tryptophan and tyrosine participated in the interaction. Synchronous fluorescence spectra revealed that the microenvironment of tryptophan in HSA turned more hydrophilic upon exposure to GNP. The α-helical content of albumin was unaltered by GNP. Approximately 37 and 23% reduction in specific activity of HSA and BSA was observed due to GNP binding. In presence of warfarin and ibuprofen the binding constants of albumin–GNP complexes were altered. A very interesting observation not reported so far is the retained antioxidant activity of albumin in presence of GNP i.e. we believe that GNPs did not bind to the free sulfhydryl groups of albumin. However enhanced levels of copper binding were observed. We have also highlighted the differential response in albumin due to gold and silver nanoparticles which could be attributed to differences in the charge of the nanoparticle.

A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.

Recombinant factor VIII Fc (rFVIIIFc) is a fusion protein consisting of a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC), we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF), with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.

Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

A Biochemical Analysis Linking APOBEC3A to Disparate HIV-1 Restriction and Skin Cancer

Human deoxycytidine deaminase APOBEC3A (Apo3A) acts as an HIV-1 restriction factor in cells of myeloid lineage yet functions separately as a potent mutator for genomic DNA. Apo3A activity and C motif deamination specificity exhibit a striking dependence on pH that reflects these two distinct biological processes. Upon infection of macrophages, HIV-1 induces the formation of autophagosomes, and requires autophagosomes for replication, whereas inhibiting lysosomal fusion indicative of late stage autophagy. Here we show that Apo3A has optimal activity and a strict 5'-YYCR motif specificity in the pH 5.8-6.1 range, characteristic of enclosed autophagosomal membrane compartments, and reflective of the mutation pattern of HIV-1. In contrast to the high activity and narrow specificity of Apo3A at acid pH, a 13-30-fold reduction in specific activity is accompanied by relaxed C deamination specificity at pH 7.4-8. Notably, Apo3A is also expressed in keratinocytes, and is up-regulated in skin lesions. At pH 7.9, we show that Apo3A generates transcription-dependent CC → TT tandem mutations on the non-transcribed strand, a hallmark signature of skin cancer. The biochemical data taken in conjunction with the biological up-regulation of Apo3A in skin lesions suggests that enzyme-catalyzed deaminations at adjacent C sites followed by normal replication generating CC → TT mutations provides an alternative molecular basis for the initiation events in skin cancer in contrast to well established pathways in which CC dimers formed in response to UV radiation either undergo nonenzymatic spontaneous deaminations or aberrant replication.

Soybean peroxidase trapped in product precipitate during phenol polymerization retains activity and may be recycled

AbstractBACKGROUNDStudies with peroxidase (EC 1.11.1.7) have demonstrated that phenolic precipitate adsorbs free enzyme, inactivating it. The end‐product inactivation model has been proposed to explain the effect. Additives, such as polyethylene glycol and Triton X‐100, were reported to effectively extend enzyme lifetime by preventing the adsorption.RESULTSIt was found that soybean peroxidase (SBP, EC 1.11.1.7) trapped in precipitate during phenol polymerization retains activity. Contrary to the end‐product inactivation model, recycling precipitate effectively utilized the active SBP. The minimum SBP concentration required for the subsequent batch reaction removal of 1 mmol L‐1 phenol from aqueous solution was reduced from 1.2 to 0.5 U mL‐1. SBP adsorption on the precipitate was proven to be reversible by the addition of Triton X‐100. Thus, a new explanation of SBP fate during the reaction is suggested: SBP is immobilized in situ in an active form with reduction of specific activity rather than inactivation. The adsorption is characterized by a Langmuir isotherm.CONCLUSIONSThe phenolic precipitate immobilizes SBP in an active form, consistent with the Langmuir isotherm model. Recycling the precipitate improves the enzyme economy in phenol removal. © 2013 Society of Chemical Industry

A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

Concentration related responses of chlorpyriphos in antioxidant, anaerobic and protein synthesizing machinery of the freshwater fish, Heteropneustes fossilis

Impacts of chlorpyriphos (2, 4, 6ppm) on the profiles of antioxidant (catalase) and anaerobic (lactate dehydrogenase) enzymes and other macromolecular contents (DNA, RNA, protein) of various tissues of the freshwater fish, Heteropneustes fossilis were studied. Chlorpyriphos significantly decreased the specific activity of catalase (CAT) and lactate dehydrogenase (LDH) in the brain, liver, gill and skeletal muscle of the fish. The reduction in specific activity might be due to the binding of chlorpyriphos or its metabolites with the enzyme molecules or affecting the synthesis and/or degradation of the enzymes. Like enzymes, the DNA, RNA and protein contents decreased in the brain, liver, gill and skeletal muscle of the fish as a function of increase in chlorpyriphos concentrations (2–6ppm). The chlorpyriphos-induced reduction in these biochemical constituents might be because of alteration in their turnover (synthesis/degradation) in different tissues. The maximum effects on CAT, LDH, DNA, RNA and protein were obtained in response to 6ppm chlorpyriphos. The present findings suggest chlorpyriphos concentration related impairment in antioxidative, anaerobic and protein synthesizing capacity of the fish. Therefore, the use of higher concentrations of chlorpyriphos should be avoided to protect the health of economically important freshwater food fish.

Ecophysiological category based toxicological responses in metabolism of earthworms: Impact of a pyrethroidal insecticide

Effects of alphamethrin (0.04 ppm, 0.08 ppm, 0.16 ppm, 0.32 ppm) on some metabolic dehydrogenases and proteins for 1, 2, 3, 4, 8 and 16 days using three ecologically different earthworm species ( Perionyx sansibaricus, Lampito mauritii and Metaphire posthuma) were studied. The first significant effect was on 2nd/3rd day and maximum response was achieved on 16th day of exposure of alphamethrin at all concentrations. Similarly, maximum effect was obtained at 0.32 ppm alphamethrin at different exposure periods. It showed a dose- and duration-dependent inhibitory effects of a pyrethroid on enzymes and proteins of earthworms. There were approximately 47%, 43% and 41% declines in specific activities of cytoplasmic malate dehydrogenase (cMDH) of P. sansibaricus, L. mauritii and M. posthuma, respectively, in response to 0.32 ppm exposure of alphamethrin for 16 days. In case of similar treatment, specific activities of mitochondrial malate dehydrogenase (mMDH) showed 57%, 51% and 45% reductions in epigeic ( P. sansibaricus), anecic ( L. mauritii) and endogeic ( M. posthuma) earthworms, respectively. Exposure of alphamethrin (0.32 ppm) also decreased the specific activities of lactate dehydrogenase (LDH) by 47% ( P. sansibaricus), 35% ( L. mauritii) and 39% ( M. posthuma) in different types of earthworms. The reductions in specific activities of a Krebs cycle enzyme (mMDH) were greater than that of a glycolytic enzyme (LDH). The protein contents declined significantly in the earthworms exposed to alphamethrin. The decreases of 41%, 36% and 30% were obtained in cytoplasmic proteins of P. sansibaricus, L. mauritii and M. posthuma, respectively. However, the mitochondrial proteins showed reductions of 45% in P. sansibaricus, 46% in L. mauritii and 38% in M. posthuma in response to alphamethrin (0.32 ppm) intoxication for 16 days. The decrease in the protein content reflected an inhibitory effect of alphamethrin on protein turnover. The most pronounced effect of alphamethrin was on metabolic enzymes and proteins of P. sansibaricus and least effect was found on M. posthuma. This clearly showed that surface dwelling species of earthworms (epigeic) are more vulnerable to toxic chemical than deep burrowing species (endogeic). Thus a possible ecophysiological link exists between the toxicological responses and ecological categories at metabolic level in tropical earthworms.

Cortical and Subcortical Connectivity Changes during Decreasing Levels of Consciousness in Humans: A Functional Magnetic Resonance Imaging Study using Propofol

While ubiquitous, pharmacological manipulation of consciousness remains poorly defined and incompletely understood (Prys-Roberts, 1987). This retards anesthetic drug development, confounds interpretation of animal studies conducted under anesthesia, and limits the sensitivity of clinical monitors of cerebral function to intact perception. Animal and human studies propose a functional "switch" at the level of the thalamus, with inhibition of thalamo-cortical transmission characterizing loss of consciousness (Alkire et al., 2000; Mashour, 2006). We investigated the effects of propofol, widely used for anesthesia and sedation, on spontaneous and evoked cerebral activity using functional magnetic resonance imaging (fMRI). A series of auditory and noxious stimuli was presented to eight healthy volunteers at three behavioral states: awake, "sedated" and "unresponsive." Performance in a verbal task and the absence of a response to verbal stimulation, rather than propofol concentrations, were used to define these states clinically. Analysis of stimulus-related blood oxygenation level-dependent signal changes identified reductions in cortical and subcortical responses to auditory and noxious stimuli in sedated and unresponsive states. A specific reduction in activity within the putamen was noted and further investigated with functional connectivity analysis. Progressive failure to perceive or respond to auditory or noxious stimuli was associated with a reduction in the functional connectivity between the putamen and other brain regions, while thalamo-cortical connectivity was relatively preserved. This result has not been previously described and suggests that disruption of subcortical thalamo-regulatory systems may occur before, or even precipitate, failure of thalamo-cortical transmission with the induction of unconsciousness.