Specific Activity Of Enzyme Research Articles

Biology, digestive enzymes and organosomatic indices of Chrysichthys nigrodigitatus (Lacépède, 1803) from Oyan Dam, Southwestern Nigeria

Some aspects of the biology, digestive enzymes and organosomatic indices of Chrysichthys nigrodigitatus purchased from fishermen in Oyan dam was investigated. This study was aimed at providing information on the composition of food materials found in the gut and specific activities of selected enzymes as it affects the domestication of the species. 100 specimens of the species were examined for stomach contents, length-weight relationship, digestive enzyme assay and organosomatic indices, using standard methods. Food items observed were detritus (4%), fish part (12%), Insecta (13%). Sand was observed to be 11% of total stomach volume. The logarithmic equation for length-weight relationship lnW=2.68lnl-3.79 indicated that an increase in length led to a corresponding increase in weight with ‘R’ = 0.611, calculated ‘r’=0.78, ‘a’=2.68 and ‘b’=3.79 indicating positive allometric growth pattern. Amylase exhibited high activity in the stomach, while lipase and proteinase in the stomach and posterior intestine. Specific activities of digestive enzymes showed significant differences (p<0.05). Viscerosomatic (2.92± 0.25), hepatosomatic (2.27± 0.22) indices and Fulton condition factor (1.93± 0.06) were recorded. Feed items present in the species confirm its overlapping feeding habit, indicating that the species is an omnivorous detritivore. This was also depicted in the activities of the different digestive enzymes.

Purification and Characterization Laccase from Trametes versicolor (L.) Lloyd in Submerged Fermentation.

<b>Background and Objective:</b> Laccase is classified as an oxidoreductase enzyme that catalyzes oxidation reactions of phenolic groups by using oxygen as its electron acceptor. Laccase isolated from <i>Trametes versicolor</i> (L.) Lloyd has a wide range of applications in the industrial sector. The use of enzymes in the industrial sector requires pure enzyme conditions from impurities so that the enzyme can maximize its ability in converting the substrate. This study aims to obtain enzyme activity and the characteristic of purified laccase enzymes isolated from <i>Trametes versicolor</i> (L.) Lloyd. <b>Materials and Methods:</b> This study was conducted with an experimental method followed by descriptive analysis. The steps of this research consist of a qualitative assay of laccase enzyme, crude laccase extract desalting by Sephadex G-25, laccase purification by Sephadex G-100 and laccase optimum pH characterization. <b>Results:</b> The result of this study showed that purification of laccase from <i>Trametes versicolor </i>(L.) Lloyd with Sephadex G-25 increases laccase enzyme-specific activity which is 10.966 U mg<sup>1</sup> and reaches 2.93-fold purity. The highest laccase enzyme activity was achieved at pH 4 with a value of laccase activity 62.39 U L<sup>1</sup>. <b>Conclusion:</b> Based on current results, purifying laccase from <i>Trametes versicolor </i>(L.) Lloyd with Sephadex G-25 was recommended which resulting higher enzyme specific activity.

How synonymous mutations alter enzyme structure and function over long timescales.

The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.

ARTP mutagenesis of phospholipase D-producing strain Streptomyces hiroshimensis SK43.001, and its enzymatic properties

Phospholipase D (PLD) is a group of enzymes that act on phospholipid molecules, which is widely used in the fields of food and medicine. PLD is extracted from animals and plants with low transesterification activity and high price. Therefore, it is benefit to screen an efficient PLD producing strain from microorganisms. A highly productive strain of PLD with transphosphatidylation activity, named Streptomyces hiroshimensis SK43.001, was screened from soil in our laboratory and mutated using atmospheric room temperature plasma (ARTP). A mutant strain SK43.001-11 with the highest enzyme activity and superior genetic stability was obtained, and its fermentation enzyme activity was 5.3 U/mL, which was 82% increased comparing to wild strain. The purification of PLD showed that the specific enzyme activity increased to 49.48 U/mg, which was 54.37-fold higher than that of the crude enzyme, with a recovery of 32.31%. In addition, enzymatic properties of PLD have revealed that the optimal pH and temperature were 7.0 and 60 °C, respectively. Metal ion Mg2+ and surfactant Triton X-100 made the enzymatic activity increased by 16% and 100%, respectively. The reaction kinetic parameters showed that the mutant PLD had higher affinity for the substrate of egg PC and better catalytic efficiency with Km, Vmax and Kcat of 30.20 mmol/L, 99.70 μmol/min and 76.33 s−1, respectively. This study may provide important inspiration for obtaining high enzyme activity strains with PLD.

Evaluating C trends in clayey Cerrado Oxisols using a four-quadrant model based on specific arylsulfatase and β-glucosidase activities

Management practices that promote the stabilization of extracellular enzymes in soil matrix, increasing their potential activity, favor the formation and stabilization of soil organic carbon (SOC). In this study, we present a four-quadrant model to evaluate C trends in clayey Cerrado Oxisols, based on the relationship between SOC and the average activities of arylsulfatase and β-glucosidase per unit of SOC (average specific enzyme activity, ASEA). This model assumes that areas under the same long-term soil use and management practices achieve a balance between ASEA and SOC. However, due to the greater sensitivity of soil enzymes, this balance is temporarily altered after the adoption of agricultural management practices that improve or degrade the overall soil quality (SQ). To calibrate the model, relationships were established between SOC and ASEA (0 to 10 cm soil samples) and the relative cumulative grain yield of soybean and corn in long-term field experiments. Threshold values for SOC and ASEA were defined as the equivalent of 50% of the maximum cumulative corn and soybean yields. These thresholds were used to divide an ASEA vs. SOC scatter plot into four quadrants. Quadrants 1 (high SOC/high ASEA) and 3 (low SOC/low ASEA) represent stable patterns of high- and low-quality soils, respectively. Quadrants 2 (low ASEA/high SOC) and 4 (high ASEA/low SOC) represent transitional patterns of soils undergoing biological degradation (C loss) and regenerative processes (C gain), respectively. A dataset containing 102 soil samples from commercial farms with contrasting management histories was used to validate the four-quadrant model, whereas a dataset of 1212 soil samples from representative Cerrado agricultural areas was used to characterize trends in soil C. The ASEA/SOC four-quadrant model provides a simple graphical representation that allows farmers to identify locations where soil management is following SOC gain or loss trajectories quickly and easily. This information can be used to make sound management decisions.

Microbicide Containing Ellagic Acid Can Inhibit HIV-1 Infection.

Ellagic acid (EA) has a wide range of biological effects. The purpose of this study was to investigate the in vitro effects of EA on HIV-1 replication, viral enzyme activity and cytokine secretion by infected cells. The anti-HIV-1 activity of EA in solution was determined in vitro using the infection of TZM-bl cells by the nano luciferase-secreting R5-tropic JRCSF strain of HIV-1, which allows for the quantification of viral growth by measuring nano luciferase in the culture supernatants. The effect of EA on the cytokine secretion of TZM-bl cells was determined by a multiplexed bead array after 48 h of HIV-1 exposure. The antiviral effect of EA in the gel formulation (Ellagel), as would be used for vaginal application, was investigated by the inhibition of infection of UC87.CD4.CCR5 cells with R5-tropic pBaLEnv-recombinant HIV-1. EA in solutions of up to 100 µM was not toxic to TZM-bl cells. EA added either 1 h before or 4 h after HIV-1 exposure suppressed the replication of R5-tropic HIV-1 in TZM-bl cells in a dose-dependent manner, with up to 69% inhibition at 50 µM. EA-containing solutions also exhibited a dose-dependent inhibitory effect on HIV-1 replication in U87 cells. When EA was formulated as a gel, Ellagel containing 25 µM and 50 µM EA inhibited HIV-1 replication in U87 cells by 56% and 84%, respectively. In assays of specific HIV-1 enzyme activity, Ellagel inhibited HIV-1 integrase but not protease. EA did not significantly modulate cytokine secretion. We conclude that EA either in solution or in a gel form inhibits HIV infection without adverse effects on target cells. Thus, gel containing EA can be tested as a new microbicide against HIV infection.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

BackgroundOur recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction.ResultsBased on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction.ConclusionThis study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

TMET-19. DEFINING A NOVEL ROLE FOR CD97 IN REGULATING GBM GLYCOLYTIC METABOLISM

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Despite multimodal therapy, disease recurrence is inevitable. To identify novel vulnerabilities of GBM, we performed an arrayed CRISPR/Cas9 screen against select adhesion G protein-coupled receptors (aGPCRs), many of which we found to be de novo expressed in GBM. Knockout of CD97, previously implicated in GBM cell migration, produced the most striking proliferative disadvantage in patient-derived GBM cultures (PDGC) among aGPCRs tested. We found high CD97 surface expression in all our PDGCs, while levels remained nearly undetectable in non-neoplastic brain cells, confirming that CD97 is de novo expressed in GBM. Upon shRNA-mediated knockdown of CD97 in PDGCs from all three TCGA transcriptional subtypes, we observed reduced proliferation, as measured by cell cycle analysis. Notably, CD97 knockdown also significantly reduced tumorsphere formation capacity as measured by limiting dilution assays; an effect that was partially rescued upon CD97 overexpression. To elucidate mechanisms of action of CD97, we performed RNA-sequencing and GO pathway enrichment analysis from PDGCs following CD97 knockdown. The top downregulated pathways involved glycolytic metabolism, specifically involving many genes relevant for glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) processing. Indeed, when we measured metabolite levels under both steady-state and flux conditions using mass spectrometry, we observed an accumulation of G6P and a depletion of most downstream glycolytic and Krebs cycle metabolites upon CD97 knockdown. Furthermore, Seahorse metabolic assays revealed deficits in both glycolytic metabolism and oxygen consumption. We aim to interrogate the activity of specific glycolytic enzymes involved in processing G6P and F6P, pinpointing how these are influenced by CD97 signaling pathways (MAPK or Akt). Overall; our studies suggest a novel role of CD97 in regulating GBM metabolism (Warburg effect), and provide a strong scientific rationale for developing biologics to target CD97 which appears to be universally and de novo expressed in GBM.

Characterization of a novel pyruvate kinase from Trichinella spiralis and its participation in sugar metabolism, larval molting and development.

Pyruvate kinase widely exists in many parasites and plays an important role in the energy production for the parasites. Pyruvate kinase might be a potential drug target for killing the parasites. The aim of the present study was to evaluate the biological characteristics and roles of T. spiralis pyruvate kinase M (TsPKM) in sugar metabolism, larval molting and development of T. spiralis. TsPKM has two functional domains of pyruvate kinase and the tertiary structure of TsPKM is tetramer which has the enzyme active site constituted by 8 amino-acid residues (Arg71, Asn73, Asp110, Phe241, Lys267, Glu269, Asp293 and Thr325). Recombinant TsPKM (rTsPKM) was expressed and purified. The rTsPKM had good immunogenicity. RT-PCR and Western blot showed that TsPKM was transcribed and expressed at various developmental stages in T. spiralis lifecycle. Immunofluorescence test showed that TsPKM was principally located in the cuticle, muscle, stichosome, intestine and the intrauterine embryos of female adults. rTsPKM catalyzed the reaction of phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to produce pyruvic acid and adenosine triphosphate (ATP). TsPKM played an important role in the metabolism and energy production of T. spiralis. After silencing of TsPKM gene by specific dsRNA-TsPKM2, protein expression and enzyme activity of TsPKM decreased by 50.91 and 26.06%, respectively. After treatment with RNAi, natural TsPKM enzyme activity, larval molting, sugar metabolism, growth and development of T. spiralis were significantly reduced. TsPKM participates in the larval molting, sugar metabolism, growth and development of T. spiralis and it might be a candidate target of therapeutic drug of trichinellosis.

The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells.

The altered activity of drug metabolism enzymes (DMEs) is a hallmark of chemotherapy resistance. Cytochrome P450s (CYPs), mainly CYP3A4, and several oxidoreductases are responsible for Phase I metabolism of doxorubicin (DOX), an anthracycline widely used in breast cancer (BC) treatment. This study aimed to investigate the role of Phase I DMEs involved in the first stages of acquisition of DOX-resistance in BC cells. For this purpose, the expression of 92 DME genes and specific CYP-complex enzymes activities were assessed in either sensitive (MCF-7 parental cells; MCF-7/DOXS) or DOX-resistant (MCF-7/DOXR) cells. The DMEs genes detected to be significantly differentially expressed in MCF-7/DOXR cells (12 CYPs and eight oxidoreductases) were indicated previously to be involved in tumor progression and/or chemotherapy response. The analysis of CYP-mediated activities suggests a putative enhanced CYP3A4-dependent metabolism in MCF-7/DOXR cells. A discrepancy was observed between CYP-enzyme activities and their corresponding levels of mRNA transcripts. This is indicative that the phenotype of DMEs is not linearly correlated with transcription induction responses, confirming the multifactorial complexity of this mechanism. Our results pinpoint the potential role of specific CYPs and oxidoreductases involved in the metabolism of drugs, retinoic and arachidonic acids, in the mechanisms of chemo-resistance to DOX and carcinogenesis of BC.

Control of parallelized bioreactors II: probabilistic quantification of carboxylic acid reductase activity for bioprocess optimization

Autonomously operated parallelized mL-scale bioreactors are considered the key to reduce bioprocess development cost and time. However, their application is often limited to products with very simple analytics. In this study, we investigated enhanced protein expression conditions of a carboxyl reductase from Nocardia otitidiscaviarum in E. coli. Cells were produced with exponential feeding in a L-scale bioreactor. After the desired cell density for protein expression was reached, the cells were automatically transferred to 48 mL-scale bioreactors operated by a liquid handling station where protein expression studies were conducted. During protein expression, the feed rate and the inducer concentration was varied. At the end of the protein expression phase, the enzymatic activity was estimated by performing automated whole-cell biotransformations in a deep-well-plate. The results were analyzed with hierarchical Bayesian modelling methods to account for the biomass growth during the biotransformation, biomass interference on the subsequent product assay, and to predict absolute and specific enzyme activities at optimal expression conditions. Lower feed rates seemed to be beneficial for high specific and absolute activities. At the optimal investigated expression conditions an activity of 1153 U mL^{-1} was estimated with a 90% credible interval of [992, 1321] U mL^{-1}. This is about 40-fold higher than the highest published data for the enzyme under investigation. With the proposed setup, 192 protein expression conditions were studied during four experimental runs with minimal manual workload, showing the reliability and potential of automated and digitalized bioreactor systems.

Effect of co-treatment of microwave and exogenous l-phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts.

Tartary buckwheat is rich in flavonoids. The application of physical processing technology and exogenous materials treatment can effectively promote grain germination and the accumulation of bioactive secondary metabolites. The content of four flavonoids, the activities of key enzymes (phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), flavonol synthase (FLS)) and the expression of key enzyme genes (FtPAL, FtCHI, FtFLS1, FtFLS2) in Tartary buckwheat sprouts treated with microwave and l-phenylalanine (l-Phe) were investigated, and the relationship between them was analyzed to explore the mechanism of promoting flavonoid accumulation, and to provide a theoretical basis for the development of functional Tartary buckwheat sprout food. Germination can promote the synthesis of flavonoids. The contents of chlorogenic acid and rutin in 7-day sprouts increased by 13 420.63% and 225.12% compared with seeds, respectively. Under the best treatment condition T3 (microwave 250 W, 90 s, 2.9 mmol L-1 L-Phe), the specific activities of PAL, CHI and FLS in 5-day-old sprouts increased by 47.84%, 53.04% and 28.02% compared with control check (CK), respectively; and the expression of FtPAL, FtCHI and FtFlS1 increased by 39.84%, 24.78% and 33.72% compared with CK, respectively. Correlation analysis showed that the content of flavonoids in Tartary buckwheat sprouts was significantly positively correlated with the specific activities of key enzymes (P < 0.01) and dynamically correlated with genes related to the synthesis of three enzymes. It suggested that microwave and l-Phe treatment may promote the synthesis of flavonoids by promoting the expression of key enzymes genes in phenylpropane metabolism and controlling the activity of key enzymes in phenylpropane metabolism. © 2022 Society of Chemical Industry.

Dietary mixed Bacillus strains promoted the growth indices, enzymatic profile, intestinal immunity, and liver and intestinal histomorphology of Nile tilapia, Oreochromis niloticus

In the current investigation, a 4-week feeding trial was carried out to study the special effects of the dietary probiotic, Sanolife® PRO-F (a combination of Bacillus subtilis 3.25 × 109 CFU/g, Bacillus licheniformis 3.50 × 109 CFU/g, and Bacillus pumilus 3.25 × 109 CFU/g) supplementation on growth performance, serum biochemical immune parameters, intestinal digestive enzymes activities, antioxidants assay, and the expression of pro-inflammatory cytokines and antioxidant-related genes with a specific reference to intestinal histomorphometry of Nile tilapia, Oreochromis niloticus, which fed with Sanolife® PRO-F at 0 (B0), 0.5 (B0.5) and 1 (B1) g kg−1 diet, respectively. At the end of the feeding trial, the achieved results revealed that Bacillus strains probiotic supplementation significantly improved the growth performance including final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed utilization when matched to the control group (p < 0.05). Concerning serum biochemical parameters, there was a significant reduction in the serum cholesterol and triglyceride levels in fish-fed Bacillus strains (p < 0.01). Serum lysozyme activity showed greater values in the case of the B0.5 and B1 groups (p < 0.01) compared with the control. No changes were estimated in the levels of glucose, hepatic (alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and renal biomarkers (Urea, Creatinine, and Uric acid) in the serum of fish-fed B0.5 and B1 diets. Additionally, the specific activities of protease, amylase, and lipase enzymes were significantly increased upon Bacillus strains՚s incorporation in O. niloticus diets. As well, the antioxidant capacity of catalase (CAT) and superoxide dismutase (SOD) enzymes in the liver and spleen were significantly increased with diminished malondialdehyde (MDA) levels. Correspondingly, the mRNA expression of the antioxidant glutathione peroxidase (GPx) enzyme in the intestinal tissue was notably increased in the control group. The length of intestinal villi, absorptive epithelium, and goblet cell counts were greater in O. niloticus fed on Bacillus strains diets. Given the obtained results, the dietary supplementation of Sanolife® PRO-F to Nile tilapia, O. niloticus at 0.5 g kg 1 and 1 g kg−1 diet can recover the growth, feed utilization, and health condition with the safety of Sanolife® PRO-F probiotic supplementation on liver and kidney function.

Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

Heterologous Expression of the Lactobacillus sakei Multiple Copper Oxidase to Degrade Histamine and Tyramine at Different Environmental Conditions.

Biogenic amines (BAs) are produced by microbial decarboxylation in various foods. Histamine and tyramine are recognized as the most toxic of all BAs. Applying degrading amine enzymes such as multicopper oxidase (MCO) is considered an effective method to reduce BAs in food systems. This study analyzed the characterization of heterologously expressed MCO from L. sakei LS. Towards the typical substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), the optimal temperature and pH for recombinant MCO (rMCO) were 25 °C and 3.0, respectively, with the specific enzyme activity of 1.27 U/mg. Then, the effect of different environmental factors on the degrading activity of MCO towards two kinds of BAs was investigated. The degradation activity of rMCO is independent of exogenous copper and mediators. Additionally, the oxidation ability of rMCO was improved for histamine and tyramine with an increased NaCl concentration. Several food matrices could influence the amine-oxidizing activity of rMCO. Although the histamine-degrading activities of rMCO were affected, this enzyme reached a degradation rate of 28.1% in the presence of surimi. Grape juice improved the tyramine degradation activity of rMCO by up to 31.18%. These characteristics of rMCO indicate that this enzyme would be a good candidate for degrading toxic biogenic amines in food systems.

ASSESSMENT OF PROTEOLYTIC ACTIVITY OF THE AQUEOUS LEAF EXTRACT OF Clerodendrum colebrookianum AND ITS EFFECT ON THE BLOOD COAGULATION CASCADE

Clerodendrum colebrookianum is a highly medicinal plant. Plant protease is one of the main components of analyzing the anticoagulant property of the plant. From the gelatin-agar plate, the technique confirms the presence of protease activity in Clerodendrum colebrookianum. Enzymatic activity and specific enzyme activity of the aqueous extract were determined using the caseinolytic method. It was seen that the enzyme activity (U) of Clerodendrum colebrookianum is 0.99±0.004 µmol of tyrosine released/ml/min. For the aqueous leaf extract of Clerodendrum colebrookianum the Clotting Time (CT), Prothrombin Time (PTT) and activated Partial Thromboplastin Time (aPTT) was determined and it was found that the plasma recalcification time increases the normal clotting time significantly at the dose of 1µG but not significant at dose 5 µg. The prothrombin time of the crude aqueous extracts of CC was observed to increase the prothrombin time significantly at the dose of 1µG. The activated partial thromboplastin time was seen to be insignificant in Clerodendrum colebrookianum. Even though Clerodendrum colebrookianum may act as a significant anticoagulant in the common and extrinsic pathways; on the intrinsic pathway of the blood coagulation cascade it may not have any effect.

Mixed grass species differ in rhizosphere microbial community structure and function response to drought compared to monocultures

Natural grassland succession is the interactions among different grass species and various environmental factors. The dynamics of soil microbial communities along succession stages has received tremendous attention. The effects of the coexistence of different grass species on the structure and function of rhizosphere soil microbial communities, however, are poorly understood, especially under drought stress conditions. Here, we investigated how different grass planting patterns and soil water content affect rhizosphere microbial community structure and function. Two typical grass species (Bothriochloa ischaemum and Stipa bungeana) on the Loess Plateau were planted in three planting systems: B. ischaemum monoculture, S. bungeana monoculture, and a mixed culture of the two species under two water regimes (optimal water: 80% field capacity [FC]; drought stress: 60% FC). Rhizosphere soil samples were collected to evaluate the rhizosphere microbial community structure (via phospholipid fatty acid [PLFA] biomarkers) and function (via six enzyme activities). The mixed culture of grass species noticeably shifted the rhizosphere microbial community structure. It caused the rhizosphere gram-positive:gram-negative PLFA ratio and fungal:bacterial PLFA ratio to increase under optimum water conditions. In comparison, drought stress caused total PLFA, bacterial PLFA, and gram-positive PLFA biomass to increase. Drought stress caused rhizosphere-specific enzyme activity to increase under monoculture treatment and rhizosphere-specific enzyme activity to decrease under mixed culture treatment. Soil water-soluble organic carbon, pH, and water-soluble organic phosphorus were the most important factors driving changes to rhizosphere microbial community composition. In comparison, pH was the predominant factor driving variation in rhizosphere specific enzyme activities. Thus, the coexistence of grass species altered the structure and function of the rhizosphere microbial community through changes in rhizosphere pH and water-soluble nutrients. In conclusion, changes to rhizodeposits under a mixed culture of grass species likely affect the structure and function of the rhizosphere microbial community. The results of the present study improve our understanding of the impacts of grass species interaction on soil biogeochemical processes under future global drought stress scenarios.

Modification and application of highly active alkaline pectin lyase

Alkaline pectate lyase has developmental prospects in the textile, pulp, paper, and food industries. In this study, we selected BacPelA, the pectin lyase with the highest expression activity from Bacillus clausii, modified and expressed in Escherichia coli BL21(DE3). Through fragment replacement, the catalytic activity of the enzyme was significantly improved. The optimum pH and temperature of the modified pectin lyase (PGLA-rep4) were 11.0 and 70 °C, respectively. It also exhibited a superior ability to cleave methylated pectin. The enzyme activity of PGLA-rep4, measured at 235 nm with 0.2% apple pectin as the substrate, was 554.0 U/mL, and the specific enzyme activity after purification using a nickel column was 822.9 U/mg. After approximately 20 ns of molecular dynamics simulation, the structure of the pectin lyase PGLA-rep4 tended to be stable. The root mean square fluctuation (RMSF) values at the key catalytically active site, LYS168, were higher than those of the wildtype PGLA. In addition, PGLA-rep4 was relatively stable in the presence of metal ions. PGLA-rep4 has good enzymatic properties and activities and maintains a high pH and temperature. This study provides a successful strategy for enhancing the catalytic activity of PGLA-rep4, making it the ultimate candidate for degumming and various uses in the pulp, paper, and textile industries.

Ultrahigh resolution MS1/MS2-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS1/data independent-MS2 method to track the simultaneous incorporation of the heavy isotopes 13C and 15N into the moieties of purine/pyrimidine nucleotides in mammalian cells. Ultrahigh resolution-MS1 resolves and counts multiple tracer atoms in intact metabolites, while data independent-tandem MS (MS2) determines isotopic enrichment in their moieties without concern for the numerous mass isotopologue source ions to be fragmented. Together, they enabled rigorous MS-based reconstruction of metabolic networks at specific enzyme levels. We have expanded this approach to trace the labeled atom fate of [13C6]-glucose in 3D A549 spheroids in response to the anticancer agent selenite and that of [13C5,15N2]-glutamine in 2D BEAS-2B cells in response to arsenite transformation. We deduced altered activities of specific enzymes in the Krebs cycle, pentose phosphate pathway, gluconeogenesis, and UDP-GlcNAc synthesis pathways elicited by the stressors. These metabolic details help elucidate the resistance mechanism of 3D versus 2D A549 cultures to selenite and metabolic reprogramming that can mediate the transformation of BEAS-2B cells by arsenite.

Protective Effect of Rutin on Triethylene Glycol Dimethacrylate-Induced Toxicity through the Inhibition of Caspase Activation and Reactive Oxygen Species Generation in Macrophages.

Rutin, also called quercetin-3-rhamnosyl glucoside, is a natural flavonol glycoside present in many plants. Rutin is used to treat various diseases, such as inflammation, diabetes, and cancer. For polymeric biomaterials, triethylene glycol dimethacrylate (TEGDMA) is the most commonly used monomer and serves as a restorative resin, a dentin bonding agent and sealant, and a bone cement component. Overall, TEGDMA induces various toxic effects in macrophages, including cytotoxicity, apoptosis, and genotoxicity. The aim of this study was to investigate the protective mechanism of rutin in alleviating TEGDMA-induced toxicity in RAW264.7 macrophages. After treatment with rutin, we assessed the cell viability and apoptosis of TEGDMA-induced RAW264.7 macrophages using an methylthiazol tetrazolium (MTT) assay and Annexin V-FITC/propidium iodide assay, respectively. Subsequently, we assessed the level of genotoxicity using comet and micronucleus assays, assessed the cysteinyla aspartate specific proteinases (caspases) and antioxidant enzyme (AOE) activity using commercial kits, and evaluated the generation of reactive oxygen species (ROS) using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay. We evaluated the expression of heme oxygenase (HO)-1, the expression of nuclear factor erythroid 2 related factor (Nrf-2), and phosphorylation of AMP activated protein kinase (AMPK) using the Western blot assay. The results indicated that rutin substantially reduced the level of cytotoxicity, apoptosis, and genotoxicity of TEGDMA-induced RAW264.7 macrophages. Rutin also blocked the activity of caspase-3, caspase-8, and caspase-9 in TEGDMA-stimulated RAW264.7 macrophages. In addition, it decreased TEGDMA-induced ROS generation and AOE deactivation in macrophages. Finally, we found that TEGDMA-inhibited slightly the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation would be revered by rutin. In addition, the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation was enhanced by rutin. These findings indicate that rutin suppresses TEGDMA-induced caspase-mediated toxic effects through ROS generation and antioxidative system deactivation through the Nrf-2/AMPK pathway. Therefore, rutin has the potential to serve as a novel antitoxicity agent for TEGDMA in RAW264.7 macrophages.