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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]

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]

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]

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]

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.

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]

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.

Background: Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet. Results: Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells. Conclusions: Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA. [ABSTRACT FROM AUTHOR]

For the past few decades continuous increase in the levels of tropospheric ozone (O 3 ) concentrations is posing to be a threat for agricultural productivity. Two high yielding tropical rice cultivars (Malviya dhan 36 and Shivani) were evaluated against different concentrations of O 3 under field conditions. Experimental design included filtered chambers, non-filtered chambers having ambient O 3 and 10 and 20 ppb elevated O 3 above the ambient. Study was conducted to assess differential response if any in induction of antioxidative defense system, genome stability, leaf proteome, yield and quality of the product in both the test cultivars. Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) were induced under ambient and elevated levels of O 3 . Native polyacrylamide gel electrophoresis (PAGE) of SOD, CAT and POD also displayed increased enzymatic activity along with associated alterations in specific isoforms. Ascorbic acid, thiols and phenolics were also stimulated at ambient and elevated O 3 . Structural alterations in DNA of rice plants due to O 3 affecting its genome template stability (GTS) was examined using RAPD technique. 2-D PAGE revealed 25 differential spots in Malviya dhan 36 and 36 spots in Shivani after O 3 treatment with reductions in RuBisCO subunits. Reductions in yield and change in the quality of grains were also noticed. [ABSTRACT FROM AUTHOR]

Highlights: [•] 44 white-rot lignocellulytic fungal strains are screened. [•] Ganoderma lucidum can grow on alkali lignin and expresses high laccase activity. [•] The G. lucidum laccase extract enhances cellulase-catalyzed lignocellulose degradation. [ABSTRACT FROM AUTHOR]

P2X4 and P2X7 receptors are ATP-gated ion channels that are co-expressed in alveolar epithelial type I cells. Both receptors are localized to the plasma membrane and partly associated with lipid rafts. Here we report on our study in an alveolar epithelial cell line of the molecular organization of P2X7R and P2X4R receptors and the effect of their knockdown. Native gel electrophoresis reveals three P2X7R complexes of ~430, ~580 and ~760 kDa. The latter two correspond exactly in size to signals of Cav-1, the structural protein of caveolae. Interestingly knockdown of P2rx7 affects protein levels, the intracellular distribution and the supramolecular organization of Cav-1 as well as of P2X4R, which is mainly detected in a complex of ~430 kDa. Our data suggest upregulation of P2X4R as a compensatory mechanism of P2X7R depletion. [ABSTRACT FROM AUTHOR]

Oxidative stress enzymes, superoxide dismutase (SOD), catalase (CAT), and ATPase, from two Gram-positive bacteria and one Gram-negative bacterium, respectively, were tested for response to the oxidative stress caused by bensulfuron-methyl (BSM). Native PAGE was used to detect the SOD isoenzyme profiles of these bacteria. All three bacteria possessed a basal level of SOD, CAT, and ATPase activity prior to being exposed to BSM. Enzyme activities changed in a BSM-concentration-dependent manner after exposure to BSM for 24h. Activity of all the enzymes was increased and reached the first activity peak after being exposed to BSM at 1 or 1.5h, respectively, then a decline occurred, and after that another simulation appeared at 9h or 14h. Only one and three detectable SOD isoenzyme bands were observed in Gram-positive strains and the Gram-negative strain, respectively. BSM could bring short-term ecotoxicity to three bacteria, but the effect of BSM was not lethal. [Copyright &y& Elsevier]

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O2 consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O2 consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin's cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by 31P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane. [ABSTRACT FROM AUTHOR]

Objective To investigate how acetamiprid, a new insecticide, affects the activity of superoxide dismutase (SOD), catalase (CAT), and ATPase and the SOD isozyme patterns in two G bacteria, E. coli K12 and Pse.FH2, and one G+ bacterum, B. subtilis. Methods The SOD, CAT, and ATPase specific activities of cell lysates were determined spectrophotometrically at 550 nm, 240 nm, and 660 nm, respectively, with kits A001, A016, and A007. SOD isozyme patterns were detected by native PAGE analysis. Results SOD and CAT activities in the tested bacteria increased significantly in a concentration-dependent manner after different concentrations of acetamiprid were applied. The activity of SOD in B. subtilis and Pse.FH2 was stimulated and reached the highest level after treatment with 100 mg/L acetamiprid for 0.5 h. For Pse.FH2, there was another stimulation of SOD activity after acetamiprid application for about 8.0 h and the second stimulation was stronger than the first. The stimulation by acetamiprid showed a relative lag for E. coli K12. Acetamiprid seemed to exhibit a similar effect on CAT activity of the two G bacteria and had an evident influence on ATPase activity in the three bacteria within a relatively short period. Only one SOD isozyme was detectable in Pse.FH2 and B. subtilis, while different isozyme compositions in E. coli could be detected by native PAGE analysis. Conclusion Acetamiprid causes a certain oxidative stress on the three bacteria which may not only elevate SOD and CAT activities but also generate new SOD isozymes to antagonize oxidative stress. However, this oxidative stress lasts for a relatively short time and does not cause a long-term damage. [ABSTRACT FROM AUTHOR]

Fibrin (Fbn) deposits are a hallmark of staphylocoagulase (SC)-positive endocarditis. Binding of the N terminus of Staphylococcus aureus SC to host prothrombin triggers formation of an active SC·prothrombin∗ complex that cleaves host fibrinogen to Fbn. In addition, the C-terminal domain of the prototypical SC contains one pseudorepeat (PR) and seven repeats (R1 → R7) that bind fibrinogen/Fbn fragment D (frag D) by a mechanism that is unclear. Here, we define affinities and stoichiometries of frag D binding to C-terminal SC constructs, using fluorescence equilibrium binding, NMR titration, alanine scanning, and native PAGE. We found that constructs containing the PR and single repeats bound frag D with K D ∼50 to 130 nM and a 1:1 stoichiometry, indicating a conserved binding site bridging the PR and each repeat. NMR titration of PR-R7 with frag D revealed that residues 22 to 49, bridging PR and R7, constituted the minimal peptide (MP) for binding, corroborated by alanine scanning, and binding of labeled MP to frag D. MP alignment with the PR-R and inter-repeat junctions identified critical conserved residues. Full-length PR-(R1 → R7) bound frag D with K D ∼20 nM and a stoichiometry of 1:5, whereas constructs containing the PR and various three repeats competed with PR-(R1 → R7) for frag D binding, with a 1:3 stoichiometry. These findings are consistent with binding at PR-R and R-R junctions with modest inter-repeat sequence variability. CD of PR-R7 and PR-(R1 → R7) suggested a disordered flexible structure, allowing binding of multiple fibrin(ogen) molecules. Taken together, these results provide insights into pathogen localization on host fibrin networks., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate "mini-G" proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners. Graphic abstract: Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats., Competing Interests: Competing interestsNone of the authors have any competing interests to disclose., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

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. [ABSTRACT FROM AUTHOR]