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Foreign amylase addition to honey in an effort to disguise diastase activity has become a widespread form of food fraud. However, since there is no report on the investigation in Japan, we investigated foreign amylases in 67 commercial honeys in Japan. First, the α-glucosidase and diastase activities of honeys were measured, which revealed that only α-glucosidase activity was significantly low in several samples. As both enzymes are secreted from honeybee glands, it is unlikely that only one enzyme was inactivated during processing. Therefore, we suspected the presence of foreign amylase. α-Amylase in honey were assigned using protein analysis software based on LC-QTOF-MS. As a result, α-amylases from Aspergillus and Geobacillus were detected in 13 and 6 out of 67 honeys, respectively. To detect foreign amylases easily, we developed a cost-effective method using native PAGE. Conventional native PAGE failed to separate the α-amylase derived from honeybee and Geobacillus. However, when native PAGE was performed using a gel containing 1 % maltodextrin, the α-amylase from honeybee did not migrated in the gel and the α-amylase could be separated from the other two α-amylases. The results from this method were consistent with those of LC-QTOF-MS method, suggesting that the novel native PAGE method can be used to detect foreign amylases. [ABSTRACT FROM AUTHOR]

The establishment of a highly sensitive method for obtaining structural information on proteins and protein complexes in vivo has long been a technological challenge in structural biology. In recent years, protein structure analysis approaches using topdown mass spectrometry, native mass spectrometry, and cross-linking mass spectrometry, among others, have been developed, and these techniques have emerged as the most promising methods for obtaining comprehensive structural information on the cellular proteome. However, information obtained by MS alone is derived mainly from protein components that are abundant in vivo, with insufficient data on low abundance components. For the detection of those low abundance components, sample fractionation prior to mass spectrometry is highly effective because it can reduce the complexity of the sample. Polyacrylamide gel electrophoresis (PAGE), which is widely used in biochemical experiments, is an excellent technique for protein separation in a simple straightforward procedure and is also a promising fractionation tool for structural proteomics. The difficulty of recovering proteins in gels has been an obstacle, thus far limiting its application to structural mass spectrometry. With the breakthrough of PEPPI-MS, an exceptionally efficient passive extraction method for proteins in gels that appeared in 2020, various PAGE-based proteome fractionation workflows have been developed, resulting in the rapid integration of structural mass spectrometry and PAGE. In this paper, we describe a simple and inexpensive PAGE-based sample preparation strategy that accelerates the broad use of structural mass spectrometry in life science research, and discuss future prospects for achieving in-depth structural proteomics using PAGE. [ABSTRACT FROM AUTHOR]

Abstract ω-Transaminases’ (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

The peculiarity of energy metabolism in helminths is the ability to undergo transition from aerobic to anaerobic under low oxygen tension. during its adult stage. Fumarate reductase and succinate dehydrogenase of mitochondria are the two enzymes responsible during this transition and adaptation to this hypoxic environment. Earlier we had reported that three species of Senna plant, S. alata, S. alexandrina and S. occidentalis altered the morphology, ionic concentration and neurotransmission of the cestode parasite Hymenolepis diminuta. The present study aimed at exploring the mechanism of leaf extracts of the three plant species of Senna on the mitochondrial activity of the parasite that chiefly involve the NADH-fumarate reductase system which is the terminal step in phosphoenolpyruvate carboxykinase succinate pathway. The structure of mitochondria was observed through electron microsopy and its density was detected through confocal microscopy, spectroflourimetry and spectrophotometry, while enzyme activities were assayed through native gel and spectrophotometric assays. Praziquantel was tested on the parasites as a reference drug to compare its effects with that of the plant extracts. The mitochondria architecture was altered, and enzymes activity decraeased by 60% in all three plant species of Senna treated parasites which suggested that these three Senna species posses potent chemotherapeutic properties. [ABSTRACT FROM AUTHOR]

BACKGROUND: Complex biotherapeutics such as non‐glycosylated antibody fragments can be easily manufactured in Escherichia coli due to advancements in recombinant DNA technology. Ranibizumab is one such example of a complex, non‐glycosylated protein used to treat age‐related macular degeneration and macular oedema. Ranibizumab, a humanized monoclonal antibody fragment, is often expressed as inclusion bodies using the E. coli expression system. In this study, we propose a novel strategy for achieving efficient expression of antibody fragments in E. coli BL21 (DE3). Here, the whole antibody fragment (heavy chain + light chain of ranibizumab) has been engineered and cloned in a pET series vector under single promoter belonging to a prokaryotic host along with an extra ribosome binding site to allow equal expression of the light and heavy chains. RESULTS: We demonstrate heterologous expression of ranibizumab with a protein concentration of 0.4 ± 0.019 mg mL–1, confirmed using reversed‐phase high‐performance liquid chromatography with the double copy clone. The reduced and refolded antibody fragment have been analytically characterized using liquid chromatography–mass spectrometry, where masses of the in‐house clone were in correspondence with marketed product. Native polyacrylamide gel electrophoresis and surface plasmon resonance (SPR) analyses were performed to confirm the formation of purified and active recombinant product. Binding kinetics to the target analyte vascular endothelial growth factor of refolded in‐house product was found to be similar to the marketed product as per SPR (12.7 nmol L−1vs. 10.4 nmol L−1). CONCLUSION: Cloning and process optimization have been performed to enhance expression yield. The proposed strategy offers an efficient approach for enhanced production of antibody fragments in microbial hosts. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

The fast-developing field of RNA nanotechnology requires the adoption and development of novel and faster computational approaches to modeling and characterization of RNA-based nano-objects. We report the first application of Elastic Network Modeling (ENM), a structure-based dynamics model, to RNA nanotechnology. With the use of an Anisotropic Network Model (ANM), a type of ENM, we characterize the dynamic behavior of non-compact, multi-stranded RNA-based nanocubes that can be used as nano-scale scaffolds carrying different functionalities. Modeling the nanocubes with our tool NanoTiler and exploring the dynamic characteristics of the models with ANM suggested relatively minor but important structural modifications that enhanced the assembly properties and thermodynamic stabilities. In silico and in vitro, we compared nanocubes having different numbers of base pairs per side, showing with both methods that the 10 bp-long helix design leads to more efficient assembly, as predicted computationally. We also explored the impact of different numbers of single-stranded nucleotide stretches at each of the cube corners and showed that cube flexibility simulations help explain the differences in the experimental assembly yields, as well as the measured nanomolecule sizes and melting temperatures. This original work paves the way for detailed computational analysis of the dynamic behavior of artificially designed multi-stranded RNA nanoparticles.

ω-Transaminases' (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract. [ABSTRACT FROM AUTHOR]

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The molecular underpinnings associated with cognitive reserve remain poorly understood. Because animal models fail to fully recapitulate the complexity of human brain aging, postmortem studies from well-designed cohorts are crucial to unmask mechanisms conferring cognitive resistance against cumulative neuropathologies. We tested the hypothesis that functionality of the SNARE protein interactome might be an important resilience factor preserving cognitive abilities in old age. Cognition was assessed annually in participants from the Rush “Memory and Aging Project” (MAP), a community-dwelling cohort representative of the overall aging population. Associations between cognition and postmortem neurochemical data were evaluated in functional assays quantifying various species of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) machinery in samples from the inferior temporal (IT, n = 154) and middle-frontal (MF, n = 174) gyri. Using blue-native gel electrophoresis, we isolated and quantified several types of complexes containing the three SNARE proteins (syntaxin-1, SNAP25, VAMP), as well as the GABAergic/glutamatergic selectively expressed complexins-I/II (CPLX1/2), in brain tissue homogenates and reconstitution assays with recombinant proteins. Multivariate analyses revealed significant associations between IT and MF neurochemical data (SNARE proteins and/or complexes), and multiple age-related neuropathologies, as well as with multiple cognitive domains of MAP participants. Controlling for demographic variables, neuropathologic indices and total synapse density, we found that temporal 150-kDa SNARE species (representative of pan-synaptic functionality) and frontal CPLX1/CPLX2 ratio of 500-kDa heteromeric species (representative of inhibitory/excitatory input functionality) were, among all the immunocharacterized complexes, the strongest predictors of cognitive function nearest death. Interestingly, these two neurochemical variables were associated with different cognitive domains. In addition, linear mixed effect models of global cognitive decline estimated that both 150-kDa SNARE levels and CPLX1/CPLX2 ratio were associated with better cognition and less decline over time. The results are consistent with previous studies reporting that synapse dysfunction (i.e. dysplasticity) may be initiated early, and relatively independent of neuropathology-driven synapse loss. Frontotemporal dysregulation of the GABAergic/glutamatergic stimuli might be a target for future drug development.

The present study deals with a current arising problem of landless farmers and a challenge in solving an age-long difficulty of silk farmers: feeding of larvae during rainy season. On feeding wet mulberry leaves, the mortality rate of larvae increases, as a result of which productivity decreases. Biogenic synthesized silver nanoparticles were found to be effective in preserving mulberry leaves at the post-harvest stage. A major threat during preservation is the decline in the protein content of leaves, putting a negative impact over productivity, as leaf protein content is a major contributor towards silk gland development. Another threat resides with the generation of excessive reactive oxygen species (ROS), causing cellular damage and thereby enhancing senescence. SDS (sodium dodecyl sulphate) gel analysis of mulberry leaf protein reflects the preservative potential of nanosilver solution. Leaves preserved in nanosilver solution showed gel banding pattern almost constant till the entire duration of preservation, i.e. for 7 days, and the banding pattern was more prominent and invariable than the banding pattern showed by leaf protein preserved in silver nitrate and distilled water. SDS banding pattern of silkworm larvae fed with nanosilver-preserved leaves appeared almost similar to that of larvae fed with fresh leaves, which also reflects a high preservative potential of nanosilver solution. Through isozyme profiling, superoxide dismutase (SOD) and catalase (CAT) activity was found to be active in both nanosilver-preserved mulberry leaves and silk gland of larvae fed with the same preserved leaves;also, this up-regulated peroxidase activity was observed in preserved leaves. Isozyme profiling reflects the presence of sufficient defensive activity to protect against damage caused by ROS accumulation. OHR-LCMS profiled proteins through string analysis detected involvement of photosynthesis-associated proteins and stress-inhibiting proteins, helping to prevent senescence and thus enhancing shelf life. [ABSTRACT FROM AUTHOR]

Proteasomes provide the main route of intracellular protein degradation. They consist of a central protease, termed the 20S proteasome, or core particle (CP), that partners with one or more regulatory complexes. The quaternary structure of the CP is conserved across all domains of life and is comprised of four coaxially stacked heptameric rings formed by structurally related α and β subunits. In eukaryotes, biogenesis of the CP is generally assumed to involve the obligate formation of α-rings. These serve as templates upon which β subunits assemble to form half-proteasomes which dimerize to give rise to CP. Here, we demonstrate the in vivo existence of an assembly-competent intermediate containing an incomplete set of both α and β subunits. The novel intermediate exhibits a precursor-product relationship with the well characterized CP assembly intermediate, the 13S. This is the first evidence that eukaryotic CP, like its archaeal and bacterial counterparts, can assemble in an α-ring independent manner. • Sub-13S complex identified as new proteasome assembly intermediate. • This complex comprises a subset of α and β subunits, but no complete α-ring. • Sub-13S converts to the 13S intermediate, bypassing need for α-ring formation. • Suggests that multiple assembly pathways derive from common evolutionary origin. [ABSTRACT FROM AUTHOR]

Induction of host resistance is commonly considered to be the most effective disease control strategy, but no cultivar has yet been bred to effectively manage tobacco bacterial wilt. An alternative tactic is to induce host-acquired resistance against the pathogen, Ralstonia solanacearum. In this study, tobacco-acquired resistance was induced by an exopolysaccharide (EPS) fructan produced by Paenibacillus kribbensis PS04, and disease severity was reduced in pot trials. The photosynthetic pigment and leaf carbohydrate contents in tobacco leaves verified that tobacco scorch symptoms disappeared under the action of EPS. Further, when the malondialdehyde (MDA) content declined by two-thirds, lipid peroxidation was eliminated. Compared to the untreated control, the contents of non-enzymatic antioxidants, ascorbate (AsA) and glutathione (GSH), rose by 69% and 64%, respectively; while the activities of defense enzymes, superoxide dismutase (SOD) and peroxidase (POD), were enhanced by 22% and 82%, respectively, at the end of leaf sampling. This evidence was further supported by native PAGE on isoforms of SOD and POD. The results of this study demonstrate that tobacco-acquired resistance can be triggered by the EPS produced by P. kribbensis PS04. [ABSTRACT FROM AUTHOR]

The paper shows the structural diversity of cytosine (C)‐rich oligodeoxynucleotides (ODNs) arising from their detail nucleotide sequence and experimental conditions. In slightly acidic solutions, the ODN nonamers with different adenine (A) and cytosine (C) sequences can adopt non‐canonical structures involving protonated bases. A distinct secondary structure formed in (C)‐rich sequences, called i‐motif (iM), consists of hemiprotonated and intercalated cytosine base pairs (C.C+). Folding and unfolding of particular structures in solutions were monitored by 1H NMR and CD spectroscopies and native polyacrylamide gel electrophoresis (PAGE), which are capable to determine their structural characteristics. Effects of sequences and their proclivity to formation of the iM on electrochemical behaviour of the ODN nonamers were studied by electrochemical methods. The LSV signals of A and C obtained from the reductive dissolution of ODN adsorption layers on a hanging mercury drop electrode were processed by elimination voltammetry with linear scan (EVLS), which revealed complex effects of the nonamer properties (namely their primary and secondary structure confirmed in solution) on their adsorption and reduction activity. [ABSTRACT FROM AUTHOR]

Many imported honeys distributed on the Polish market compete with local products mainly by lower price, which can correspond to lower quality and widespread adulteration. The aim of the study was to compare honey samples (11 imported honey blends and 5 local honeys) based on their antioxidant activity (measured by DPPH, FRAP, and total phenolic content), protein profile obtained by native PAGE, soluble protein content, diastase, and acid phosphatase activities identified by zymography. These indicators were correlated with standard quality parameters (water, HMF, pH, free acidity, and electrical conductivity). It was found that raw local Polish honeys show higher antioxidant and enzymatic activity, as well as being more abundant in soluble protein. With the use of principal component analysis (PCA) and stepwise linear discriminant analysis (LDA) protein content and diastase number were found to be significant (p < 0.05) among all tested parameters to differentiate imported honey from raw local honeys.

Foreign amylase addition to honey in an effort to disguise diastase activity has become a widespread form of food fraud. However, since there is no report on the investigation in Japan, we investigated foreign amylases in 67 commercial honeys in Japan. First, the α-glucosidase and diastase activities of honeys were measured, which revealed that only α-glucosidase activity was significantly low in several samples. As both enzymes are secreted from honeybee glands, it is unlikely that only one enzyme was inactivated during processing. Therefore, we suspected the presence of foreign amylase. α-Amylase in honey were assigned using protein analysis software based on LC-QTOF-MS. As a result, α-amylases from Aspergillus and Geobacillus were detected in 13 and 6 out of 67 honeys, respectively. To detect foreign amylases easily, we developed a cost-effective method using native PAGE. Conventional native PAGE failed to separate the α-amylase derived from honeybee and Geobacillus . However, when native PAGE was performed using a gel containing 1 % maltodextrin, the α-amylase from honeybee did not migrated in the gel and the α-amylase could be separated from the other two α-amylases. The results from this method were consistent with those of LC-QTOF-MS method, suggesting that the novel native PAGE method can be used to detect foreign amylases., Competing Interests: The authors declare no conflicts of interest associated with this manuscript., (2023 by The Japanese Society of Applied Glycoscience.)

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state. Unlabelled Image • Styrene maleic acid lipid particles (SMALPs) can be used with native gel electrophoresis (SMA-PAGE) to separate membrane complexes • This separation method provides an excellent measure of protein quaternary structure • SMA-PAGE is complementary to immunoblotting • The lipid environment surrounding the protein(s) can be identified using mass spectrometry • Intact membrane protein-SMALPs extracted from gel can be visualised using electron microscopy [ABSTRACT FROM AUTHOR]

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools. [Display omitted] • Biliverdin-binding AnPixJg2 variant, AnPixJg2_BV4 H318 was biochemically analyzed. • AnPixJg2_BV4 H318 exhibits surface charge change upon photoconversion. • Region of the conformational change was revealed upon photoconversion. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) remains a deadly infectious disease despite all the efforts to reduce its incidence. Spread of multidrug resistant TB has seriously undermined the efforts to control the disease globally. In this study protein expression profile of MDR and sensitive isolates of MTB were analyzed and compared in order to identify proteins, which could be used in prevention, diagnosis and treatment. Methods: A sensitive and MDR isolate of Mycobacterium tuberculosis (MTB) were cultured on Middlebrook 7H9 medium and the whole cell lysates were subjected to native polyacrylamide gel electrophoresis (NPAGE) for protein expression profiling. Protein bands present in the MDR cell lysate that were not detected in the sensitive cell lysate were sent for identification by Matrix-assisted laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF-MS). Results: Comparison of the protein expression profiles showed 6 bands that were not detected in the sensitive isolates. MTB Structural Annotation database search of the mass spectrometry results identified these bands as Rv3597c, Rv0379,Rv3614c, Rv0475, Rv0462, andRv0147and global transcriptional regulation, involvement in cell wall and cell processes and intermediary metabolism and respiration were the functions attributed to these proteins. Conclusion: Our results highlighted the complexities of linking protein expression to MDR phenotype as none of the proteins identified could be linked directly to drug resistance. The proteins identified in the present study were mostly those essential for survival or virulence of the bacteria, and could be used for diagnosis or as candidate vaccine, but with a better understanding of the function of these proteins their association with the MTB resistance to antibiotics might become clear.

Polysialic acid (polySia/PSA) is an anionic glycan polymer of sialic acid, and it mostly modifies the neural cell adhesion molecule (NCAM) in mammalian brains. Quality and quantity of the polySia of the polySia–NCAM is spatio-temporally regulated in normal brain development and functions, and their impairments are reported to be related to diseases, such as psychiatric disorders and cancers. Therefore, precise understanding of the state of polySia–NCAM structure would lead to the diagnosis of diseases for which their suitable evaluation methods are necessary. In this study, to develop these evaluation methods, structures of polySia–NCAM from mouse brains at six different developmental stages were analyzed by several conventional and newly developed methods. Integrated results of these experiments clearly demonstrated the existence of different types of polySia–NCAMs in developing brains. In addition, combinational analyses were shown to be useful for precise understanding of the quantity and quality of polySia, which can provide criteria for the diagnosis of diseases.

Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches.

The molecular underpinnings associated with cognitive reserve remain poorly understood. Because animal models fail to fully recapitulate the complexity of human brain aging, postmortem studies from well-designed cohorts are crucial to unmask mechanisms conferring cognitive resistance against cumulative neuropathologies. We tested the hypothesis that functionality of the SNARE protein interactome might be an important resilience factor preserving cognitive abilities in old age. Cognition was assessed annually in participants from the Rush “Memory and Aging Project” (MAP), a community-dwelling cohort representative of the overall aging population. Associations between cognition and postmortem neurochemical data were evaluated in functional assays quantifying various species of the SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) machinery in samples from the inferior temporal (IT, n  = 154) and middle-frontal (MF, n  = 174) gyri. Using blue-native gel electrophoresis, we isolated and quantified several types of complexes containing the three SNARE proteins (syntaxin-1, SNAP25, VAMP), as well as the GABAergic/glutamatergic selectively expressed complexins-I/II (CPLX1/2), in brain tissue homogenates and reconstitution assays with recombinant proteins. Multivariate analyses revealed significant associations between IT and MF neurochemical data (SNARE proteins and/or complexes), and multiple age-related neuropathologies, as well as with multiple cognitive domains of MAP participants. Controlling for demographic variables, neuropathologic indices and total synapse density, we found that temporal 150-kDa SNARE species (representative of pan-synaptic functionality) and frontal CPLX1/CPLX2 ratio of 500-kDa heteromeric species (representative of inhibitory/excitatory input functionality) were, among all the immunocharacterized complexes, the strongest predictors of cognitive function nearest death. Interestingly, these two neurochemical variables were associated with different cognitive domains. In addition, linear mixed effect models of global cognitive decline estimated that both 150-kDa SNARE levels and CPLX1/CPLX2 ratio were associated with better cognition and less decline over time. The results are consistent with previous studies reporting that synapse dysfunction (i.e. dysplasticity) may be initiated early, and relatively independent of neuropathology-driven synapse loss. Frontotemporal dysregulation of the GABAergic/glutamatergic stimuli might be a target for future drug development. [ABSTRACT FROM AUTHOR]

Spodoptera litura (Fabricius) is a widely distributed worldwide insect pest, particularly of vegetable crops. To determine the sensitivity of S. litura to insecticides, the toxicities of beta-cypermethrin, chlorpyrifos, emamectin benzoate, methoxyfenozide, and indoxacarb were tested from 2014 to 2016 using a diet incorporation assay on populations of S. litura collected from different districts in Sichuan Province and a susceptible strain (Lab-HN). Compared to the Lab-HN strain, field populations showed high levels of resistance to beta-cypermethrin (267.3- to 1789.2-fold). Field populations exhibited low or moderate resistance to chlorpyrifos in 2014 (8.9- to 24.4-fold) and high levels in 2016 (440.5- to 950.5-fold). All populations collected in 2014 and 2016 were susceptible or had a low or moderate level of resistance to indoxacarb (2.2- to 31.0-fold). No field populations exhibited resistance to emamectin benzoate or methoxyfenozide (0.7- to 1.5-fold and 0.9- to 1.4- fold) in 2014, whereas the resistance in 2016 increased to 29.8- to 55.2-fold and 38.1- to 59.0- fold, respectively. A synergist experiment indicated that the Lab-HN strain and field-collected population from Shuangliu in 2016 (SL16 population) to beta-cypermethrin may be related to cytochrome P450 monooxygenases (P450s), with the highest synergism ratios (SR) of piperonyl butoxide (PBO) of 34.4- and 73.3-fold, but not to carboxylesterase (CarE) or glutathione-S- transferase (GST). We detected the activities of detoxification enzymes and found that the ethoxycoumarin O -deethylase activities were also the strongest in the Lab-HN strain and the SL16 population with the highest SR values of 2.6- and 4.1-fold, respectively. Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that the inhibition of in vivo P450 activity caused by PBO was due to the binding of the synergist to the E5 or E6 isozyme, which were absent in the Lab-HN strain. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CEs) are α,β-hydrolase fold proteins that catalyse the hydrolysis of a wide range of endogenous and exogenous compounds. In mammals, these enzymes are involved in the detoxification of certain pesticides and drugs. However, this toxicological role of CEs has received little attention in marine organisms such as the mussel Mytilus galloprovincialis . Therefore, the purpose of this study was to examine whether mussel CE activity is sensitive to inhibition by environmental chemicals of current concern (human pharmaceuticals and personal care products or PPCPs; i.e fluoxetine, loperamide, simvastatin, fenofibrate, nonylphenol and triclosan), so this chemical interaction may be considered as a detoxification mechanism comparable to that of organophosphorus pesticides. First, we examined the basal levels of CE activity in multiple tissues of M. galloprovincialis , using a wide range of colorimetric substrates, i.e., p -nitrophenyl acetate (pNPA), p -nitrophenyl butyrate (pNPB), 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB) and 2-naphtyl acetate (2-NA). Second, we tested for a substrate-dependence of detecting CE inhibition using the model organophosphorus dichlorvos. Finally, some PPCPs were tested for in vitro CE inhibition using multiple substrates. Results showed that long-chain esters (pNPB and 1-NB) provided the highest CE-mediated hydrolysis rates compared with pNPA or 1-NA. Moreover, the digestive gland and gills displayed the highest CE activities compared with haemolymph. As expected, the esterase enzyme was very sensitive to dichlorvos (IC50 s in the nM range), but dose-inhibition relationships were markedly dependent on the type of substrate used for enzyme assay. The in vitro inhibition kinetics identified triclosan as a potential CE inhibitor (IC50 = 7.07–27.2 μM for digestive gland, IC50 = 10.8–104 μM for gill CE activity), although other PPCPs such as simvastatin, fenofibrate and nonylphenol also decreased the enzyme activity to a lesser extent. In-gel esterase staining after non-denaturing polyacrylamide gel electrophoresis confirmed the inhibitory effect of triclosan upon CE activity, which was more pronounced with the substrate 1-NB. These findings suggest that CE inhibition may be a suitable biomarker of PPCP exposure to be incorporated into the battery of sub-individual indicators of PPCP exposure and toxicity. However, the selection of appropriate substrates is a key issue. Our results indicated that pNPB and 1-NB are the most suitable reporters for detecting inhibition of CE by both organophosphorus and PPCPs in mussels. [ABSTRACT FROM AUTHOR]

Photosynthetic organisms rapidly adjust the capture, transfer and utilization of light energy to optimize the efficiency of photosynthesis and avoid photodamage. These adjustments involve fine-tuning of expression levels and mutual interactions among electron/proton transfer components and their associated light-harvesting antenna. Detailed studies of these interactions and their dynamics have been hindered by the low throughput and resolution of currently available research tools, which involve laborious isolation, separation and characterization steps. To address these issues, we developed an approach that measured multiple spectroscopic properties of thylakoid preparations directly in native polyacrylamide gel electrophoresis gels, enabling unprecedented resolution of photosynthetic complexes, both in terms of the spectroscopic and functional details, as well as the ability to distinguish separate complexes and thus test their functional connections. As a demonstration, we explore the thylakoid membrane components of Chlamydomonas reinhardtii acclimated to high and low light, using a combination of room temperature absorption and 77K fluorescence emission to generate a multi-dimensional molecular and spectroscopic map of the photosynthetic apparatus. We show that low-light-acclimated cells accumulate a photosystem I-containing megacomplex that is absent in high-light-acclimated cells and contains distinct Lhc II proteins that can be distinguished based on their spectral signatures. [ABSTRACT FROM AUTHOR]

Ascochyta pisi induces leaf spot in Pisum sativum . Currently A . pisi was found pathogenic against 4 pea varieties. Ten concentrations of Eucalyptus camaldulensis leaf extract and its 7 fractionations were appraised for antifungal activity against A . pisi . The highest concentration revealed 78% suppression in growth. Fractionation was carried out successively with n-hexane, Chloroform, Ethyl acetate and n-Butanol. In in vivo bioassays, all the solvent fractions particularly n-Butanol controlled pathogen efficiently on all varieties. In RT-PCR, all genes were expressed in control and treated plants of Greenfeast but the intensity of gene 2 and 8 was upregulated in treated plants. n-Butanol induced the over expression of gene 3 and 7 in Meteor. Then, in native PAGE Glu 1 was the glucanase isozyme, detected only in variety Greenfeast. Control plants of Rondo showed Glu 2 and 3, while its treated plants had Glu 1, Glu 2, Glu 3 and Glu 4. [ABSTRACT FROM AUTHOR]

Preparation of modified and hybrid ferritin provides a great opportunity to understand the mechanisms of iron loading/unloading, protein self-assembly, size constrained nanomaterial synthesis and targeted drug delivery. However, the large size (M.W. = 490 kDa) has been limiting the separation of different modified and/or hybrid ferritin nanocages from each other in their intact assembled form and further characterization. Native polyacrylamide gel electrophoresis (PAGE) separates proteins on the basis of both charge and mass, while maintaining their overall native structure and activity. Altering surface charge distribution by substitution of amino acid residues located at the external surface of ferritin (K104E & D40A) affected the migration rate in native PAGE while internal modification had little effect. Crystal structures confirmed that ferritin nanocages made up of subunits with single amino acid substitutions retain the overall structure of ferritin nanocage. Taking advantage of K104E migration behavior, formation of hybrid ferritins with subunits of wild type (WT) and K104E were confirmed and separated in native PAGE. Cage integrity and iron loading ability (ferritin activity) were also tested. The migration pattern of hybrid ferritins in native PAGE depends on the subunit ratio (WT: K104E) in the ferritin cage. Our work shows that native PAGE can be exploited in nanobiotechnology, by analyzing modifications of large proteins like ferritin. Significance Native PAGE, a simple, straight-forward technique, can be used to analyze small modification (by altering external surface charge) in large proteins like ferritin, without disintegrating its self-assembled nanocage structure. In doing so, native PAGE can complement the information obtained from mass spectrometry. The confirmation and separation of modified and hybrid ferritin protein nanocages in native PAGE, opens up various prospects of bio-conjugation, which can be useful in targeted drug delivery, nanobiotechnology and in understanding nature's idea of synthesizing hybrid ferritins in different human tissues. [ABSTRACT FROM AUTHOR]

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.

The response to arsenic was evaluated for photosynthetic pigments, lipid peroxidation and anti-oxidative enzymes in black gram. Black gram (Vigna mungo) subjected to arsenic doses of 100 µM and 200 µM, showed increased amount of lipid peroxidation. Activity of peroxidase (POD) increased tremendously in arsenic treatedVignaleaves over control. Similarly, activities of enzymes like superoxide dismutase (SOD) and ascorbate peroxidase (APX) also increased with increase in arsenic. Analysis of native PAGE superoxide dismutase activity showed one manganese (Mn)-SOD and two copper/zinc (Cu/Zn)-SOD isoenzymes in black gram leaves, whose intensity increased especially at 200 µM arsenic treatment. However, activity of catalase (CAT) decreased with increase in concentrations of arsenic. This clearly indicates that except CAT, all studied antioxidative enzymes like SOD, POD and APX showed increased expression at arsenic treatments, which reflects their protective role against arsenic toxicity in black gram plants. Photosynthetic pigments like chlorophyll and carotenoids showed reduction with increasing concentrations of arsenic in treatment solutions. [ABSTRACT FROM PUBLISHER]

In addition to their role in correctly attaching specific amino acids to cognate tRNAs, aminoacyl-tRNA synthetases (aaRS) have been found to possess many alternative functions and often bind to and act on other nucleic acids. In contrast to the well-defined 3D structure of tRNA, the structures of many of the other RNAs recognized by aaRSs have not been solved. Despite advances in the use of X-ray crystallography (XRC), nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM) for structural characterization of biomolecules, significant challenges to solving RNA structures still exist. Recently, small-angle X-ray scattering (SAXS) has been increasingly employed to characterize the 3D structures of RNAs and RNA-protein complexes. SAXS is capable of providing low-resolution tertiary structure information under physiological conditions and with less intensive sample preparation and data analysis requirements than XRC, NMR and cryo-EM. In this article, we describe best practices involved in the process of RNA and RNA-protein sample preparation, SAXS data collection, data analysis, and structural model building. [ABSTRACT FROM AUTHOR]

Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the α and β subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo . Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo . [ABSTRACT FROM AUTHOR]

In-gel hydrolysis of para-nitrophenyl phosphate (p -NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with K m , V max and k cat values of (0.7 ± 0.02) mM, (80 ± 4.5) μmol min−1 and (39.2 ± 2.2) × 104 s−1 respectively for p -NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO 4 , Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO 4 and 65% activity at 3 mM Hg2+ showing it's PO 4 3− irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp -ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and β-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and βME reduce the C308–C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase. [Display omitted] • Zymography used as a guide for purification, characterization and inhibition analysis of 54 kDa alkaline phosphatase from a Hg2+ tolerant K. pneumonia e. • Zymography reveal that thiohydroxyals reduce it's C308–C358 disulfide bond resulting in an open conformation and loss of activity. • Hg2+ inhibits the enzyme by binding to the surface-accessible metal binding motifs without changing it's conformation. [ABSTRACT FROM AUTHOR]