Determination Of Enzyme Activity Research Articles

The use of isothermal titration calorimetry for the assay of enzyme activity: Application in higher education practical classes.

Determination of enzyme activity is crucial for discovery, research, and development in life sciences. The activity of enzymes is routinely determined using spectrophotometric assays that measure rates of substrate consumption or product formation. Though colorimetric-based detection systems are simple, rapid, and economical to perform, the majority of enzymes are unsuitable for this technique as their substrates/products do not absorb in the UV or visible range. This limitation can be addressed by the use of coupled-enzyme assays or artificial chromogenic substrates; however these approaches have their own drawbacks. Here, we describe a method based on the use of an isothermal titration calorimeter (ITC) to measure the heat produced or absorbed during any enzyme-catalyzed reaction. The concept of calorimetric enzyme assays was demonstrated for the determination of enzyme hexokinase activity, which cannot be monitored colorimetrically without first coupling it to another enzymatic reaction. The assay is suitable for incorporation into undergraduate laboratory classes, providing students with an appreciation for; the versatility and ease of use of ITC assays; ITC as a flexible generic method for exploring the functional characteristics of uncharacterized enzymes; an activity detection parameter suitable for enzymes that either have no straightforward colorimetric methods available or require the use of nonartificial chromogenic substrates.

THE INFLUENCE OF NON-PROTEIN AMINO ACIDS AND PEPTIDES ON BACTERIAL COLLAGENASE

Bacterial collagenolytic proteases have drawn increasing attention due to their essential role in the global nitrogen cycling and their virulent role in some diseases. The inhibition of these enzymes is attractive, as it does not attack the pathogen directly but rather blocks the colonization and infiltration of the host by the clostridia. Optically active non-protein α-amino acids have been screened for their ability to inhibit collagenase of Clostridium histolyticum. Both structure-based drug design approach (modeling) and that of determining enzyme activity in the presence of amino acids have been used to identify low molecular weight inhibitors of collagenase. The compounds able to inhibit collagenase activity have been revealed and IC50 values have been estimated.

Sialylated glycan-modulated biomimetic ion nanochannels driven by carbohydrate–carbohydrate interactions

Ion channels play key roles in regulating the ion environment inside and outside the cell. Sialylated glycans (SGs) at the terminus of voltage-gated ion channels (VGICs) are abundant and directly control the switch of VGICs, while SGs on the cell surface are also closely related to virus infection, tumor growth, and metastasis. Here, we report a biomimetic ion nanochannel device that can be precisely regulated by SG. The nanochannel device is composed of a chemically etched polyethylene terephthalate film featuring conical nanochannels and a polyethyleneimine-g-malcopyranoside (abbreviated to Mal-PEI). Maltose, core-binding units in Mal-PEI, forms multiple hydrogen-bonding interactions with SG, which triggers globule-to-coil transition of the polymer chain and blocks transmembrane ion transport, resulting in a remarkable decrease in the ionic current of the nanochannel. Based on the changes in the ionic current, this device can precisely discriminate α2-3- and α2-6-linked sialyllactose, as well as SGs and neutral saccharides. Importantly, the nanochannel device can monitor the sialylation process of lactose catalyzed by α2,6-sialyltransferase in real time, showing its good potential in enzyme activity determination and in vitro enzyme identification. This work constructs an SG-modulated nanochannel with selective and smart ion-gating behavior, exhibiting unique advantages in SG responsiveness and enzymatic activity monitoring.

Isolation and Nitrogen Removal Efficiency of the Heterotrophic Nitrifying-Aerobic Denitrifying Strain K17 From a Rare Earth Element Leaching Site.

K17, an indigenous and heterotrophic nitrifying-aerobic denitrifying bacterium, was isolated from the soil of a weathered crust elution-deposited rare earth ore leaching site in Longnan County, China. Strain K17 was identified as Pseudomonas mosselii. In this study, the morphological characteristics of strain K17 were observed and the optimal ammonia nitrogen removal conditions for the strain were studied using a single-factor experiment. Key enzyme activities were determined, and we also explored the ammonia nitrogen removal process of strain K17 on simulated leaching liquor of the rare earth element leaching site. Based on the determination of ammonia nitrogen removal and enzyme activity, it was found that strain K17 has both heterotrophic nitrifying and aerobic denitrifying activities. In addition, single-factor experiments revealed that the most appropriate carbon source for strain K17 was sodium citrate with a C/N ratio of 10 and an initial NH4+-N concentration of 100 mg/l. Furthermore, the optimal initial pH and rotation speed were 7 and 165 r/min, respectively. Under optimal conditions, the ammonia nitrogen removal efficiency of strain K17 was greater than 95%. As an indigenous bacterium, strain K17 has great potential for treating residual ammonium leaching solutions from rare earth element leaching sites.

Effect of indaziflam on microbial activity and nitrogen cycling processes in an orchard soil

Indaziflam is a preemergent herbicide widely used for the control of weeds in pecan ( Carya illinoinensis ) orchards in the US southwestern region. Given the paucity of data regarding indaziflam's impact on the biochemical properties of soils supporting pecan production, this study was conducted to evaluate the effect of different application rates of indaziflam on soil microbial activity, diversity and biochemical processes related to nitrogen cycling. During 2 consecutive growing seasons (2015 and 2016), soil samples were obtained from experimental mesocosms consisting of soil-filled pots where pecan saplings were grown, and that were treated with indaziflam applied at 2 different rates (25 and 50 g ai ha -1 ). Soil samples were collected one week before and one week after herbicide application, and used for determination of soil microbial biomass, microbial diversity, nitrogen (N) mineralization and β-glucosaminidase enzyme activity. Soil samples collected from control mesocosms were treated in the laboratory with 2 rates of indaziflam (75 and 150 g ai ha -1 ) to determine immediate impact on microbial activity. No significant effect of herbicide treatment on soil respiration and microbial biomass was detected. Results showed a slight to moderate decrease in microbial diversity (7 % in 2015, 44 % in 2016). However, decreased β-glucosaminidase activity with herbicide treatment was observed with soils from the mesocosms (33% decline) and soils treated with indaziflam in the laboratory (45 % decline). The mineral N pool was generally dominated by NH 4 + after indaziflam application, an observation consistent with the drastic decrease (75 %) in nitrification activity measured in the laboratory experiment. This study results indicate that indaziflam, even when applied at higher than recommended rates, has limited effects on total soil microbial activity but could affect N cycling processes.

Subchronic Toxicity of Microcystin-LR on Young Frogs (Xenopus laevis) and Their Gut Microbiota.

Although toxic effects of microcystins (MCs) in mammals and fish have been extensively studied, the effects of MCs on the immune system and gut microbiota of amphibians have not received sufficient attention. As MCs cause general damage to the vertebrate liver and immune system and trigger an inflammatory response, and the gut microbiota is closely related to host metabolism and immunity, we speculated that MCs can cause changes in the immune system and gut microbiota of amphibians. To verify this, we examined the intestinal and liver injury of Xenopus laevis exposed to different microcystin-leucine-arginine (MC-LR) concentrations and the effects on the gut microbiota through high-throughput sequencing of 16S rDNA of the gut microbiota combined with histopathological analysis, enzyme activity determination, and qRT-PCR. Our results showed that MC-LR caused focal infiltration of inflammatory cells and increased the number of T cells and local congestion and vacuolization in X. laevis liver, but reduced the number, density, height, and regularity of villi. These liver and intestinal injuries became more obvious with an increase in MC-LR concentration. MC-LR significantly decreased the activities of malondialdehyde and alkaline phosphatase and the expression of TGF-β in the liver. Moreover, MC-LR significantly altered the gut microbiota of X. laevis. The relative abundance of Firmicutes and Bacteroidetes in high-concentration MC-LR groups was significantly reduced compared to that in low-concentration MC-LR groups, whereas Fusobacteria was significantly enriched. The metabolic gene composition of the gut microbiota in low-concentration MC-LR (≤5 μg/L) groups was significantly different from that in high-concentration MC-LR (≥20 μg/L) groups. These results deepen our understanding of the toxicity of MCs to aquatic organisms and assessment of the ecological risk of MCs in amphibians.

Resistance of Sogatella furcifera to triflumezopyrim mediated with the overexpression of CYPSF01 which was regulated by nuclear receptor USP

Sogatella furcifera is one of the main agricultural pests in many Asian countries, bringing about enormous injury. A triflumezopyrim-resistant (Tri) strain of S. furcifera was established through continuous screening in laboratory. The determination of synergist and enzyme activity indicated that P450s, especially for the upregulation expression of CYPSF01, played a key role in the increased resistance, confirmed by RNAi, and the recombinant protein of CYPSF01 and NADPH-P450 reductase was able to degrade triflumezopyrim. CYPSF01 had an obviously co-expression relationship with nuclear receptor ultraspiracle (USP), which were all significantly up-regulated when exposed to triflumezopyrim. Further, a USP-binding motif MA0534.1 was enriched from the upregulated peaks by Assay for Transposase Accessible Chromatin (ATAC-seq) analysis, which exited in the peaks located on the promoter of CYPSF01; the yeast one-hybrid experiments confirmed that USP could bind to the CYPSF01 promoter. And the USP interference significantly down-regulated CYPSF01 expression, and resulted in the significantly increasing sensitivity to triflumezopyrim, its mortality rate increased 28.37%. Therefore, the overexpression of USP could cause to the overexpression of CYPSF01, ultimately resulting in the resistance to triflumezopyrim in S. furcifera.

Characterization of Two Wheat-Derived Glycoside Hydrolase Family-10 Xylanases Resistant to Xylanase Inhibitors

Xylanase inhibitors inhibit the activities of microbial xylanases and seriously compromise the efficacy of microbial xylanases added to modify cereals. Cereal endogenous xylanases are unaffected by these xylanase inhibitors, but little information is available regarding their effects in improving cereal quality, a neglected potential application. As a strategy for circumventing the negative effects of xylanase inhibitors, the objective of this study was to use genetic engineering to obtain sufficient amounts of active endo-1,4-β-D-xylanase from wheat to analyze the characteristics of its structure. The endo-1,4-β-D-xylanase from wheat was heterologously expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, MALDI-TOF/TOF (MS) analyses, and enzyme activity determination confirmed 2 active endo-1,4-β-D-xylanases (EXY3 and EXY4) were successfully obtained. The molecular weights (MW) and isoelectric point (pI) of EXY3 were 36.108 kDa and 5.491, while those of the EXY4 protein were 41.933 kDa and 5.726. They both contained the same catalytic domain of GH10 xylanases from G266 to V276 and have the same catalytic site, Glu273. They shared the same putative N-glycosylation sites (N62-T63-S64 and N280–V281–S282) and 3 putative O-glycosylation sites (Ser8, Ser9, and Thr21), but EXY4 had an additional O-glycosylation site (Thr358). EXY3 was smaller than EXY4 by 51 amino acids because of a nonsense mutation and premature termination. They both had the 8-fold beta/alpha-barrel (TIM-barrel) fold. The specific activities of EXY3 and EXY4 were 152.0891 and 67.2928 U/mg, respectively. This work demonstrates a promising way to obtain wheat xylanases by genetic engineering; the properties of the enzymes indicate their potential application in cereal-based industries.

Influence of three ultrasound treatments on viability, culturability, cell architecture, enzymatic activity and metabolic potential of Lacticaseibacillus paracasei 90

The effects of ultrasonic bath (UB) and ultrasonic probe (UP) treatments on culturability, viability, cell architecture, and enzymatic activity of Lacticaseibacillus paracasei 90 (L90) were studied by cell count, flow cytometry, scanning electron microscopy (SEM) and enzyme activity determinations. To evaluate the metabolic potential of the treated cells, milk was inoculated with treated or untreated bacteria, and incubated for 20 h at 37 °C. SEM revealed cellular damage in some UP-treated cells; flow cytometry and enzyme activity analysis reflected an overall increase in permeability and lysis. UB increased the proteolytic activity of L90 and reduced its ability to produce acid. The damage of the cellular integrity caused by UP led to an increase in enzyme accessibility to the substrate, positively correlated with enhanced diacetyl and acetoin production. Depending on equipment used and exposure time, ultrasonic waves may be used to enhance the metabolic potential of starter and adjunct cultures.

A new method for determining of the effectiveness of feed enzymes

Recently, there is no single universally recognized and standard method for determining the activity of enzymes that destroy non-starchy polysaccharides (NPS). Each manufacturer of NPS enzymes uses its own approach, as well as its own analytical conditions (pH, temperature, substrate, etc.). In practice, the determination of enzyme activity shows the activity of a specific enzyme that is part of a commercial product. This creates difficulties for consumers in orienting and comparing this variety of available products on the market. In this paper Scientific and Research Center “Cherkizovo” proposed to use the concept of “enzyme efficiency” and developed a new method for its analysis. It has been demonstrated that the final product of the enzymatic reaction can act as a working indicator of the efficiency of the enzyme. As a result, it has been shown that different enzyme complexes contribute to the different release of free simple sugars in an aqueous colloidal solution of crushed grain incubated at a temperature of 39 °C with the efficiency of 6 to 160 % compare with control. For example, in wheat the increase in the release of sugars (i.e. efficiency of multi enzyme mixtures) it ranged from 22 to 76 %, in barley it was from 11 to 160 %, in corn it was from 6 to 74 %, in grain mixtures it was from 18 to 60 %. The authors demonstrate the simple way to evaluate the effectiveness of enzymes and suggest using it as a preliminary result when evaluating the possibility of selecting the effective multi enzyme composition for a specific diet and a specific feedstock, including non-traditional. This will allow a preliminary evaluation of the effectiveness of feed enzymes for specific raw materials and diets.

Gene therapy of yeast NDI1 on mitochondrial complex I dysfunction in rotenone-induced Parkinson\u2019s disease models in vitro and vivo

PurposeParkinson's disease (PD) is the second most common neurodegenerative disease without cure or effective treatment. This study explores whether the yeast internal NADH-quinone oxidoreductase (NDI1) can functionally replace the defective mammalian mitochondrial complex I, which may provide a gene therapy strategy for treating sporadic PD caused by mitochondrial complex I dysfunction.MethodRecombinant lentivirus expressing NDI1 was transduced into SH-SY5Y cells, or recombinant adeno-associated virus type 5 expressing NDI1 was transduced into the right substantia nigra pars compacta (SNpc) of mouse. PD cell and mouse models were established by rotenone treatment. The therapeutic effects of NDI1 on rotenone-induced PD models in vitro and vivo were assessed in neurobehavior, neuropathology, and mitochondrial functions, by using the apomorphine-induced rotation test, immunohistochemistry, immunofluorescence, western blot, complex I enzyme activity determination, oxygen consumption detection, ATP content determination and ROS measurement.ResultsNDI1 was expressed and localized in mitochondria in SH-SY5Y cells. NDI1 resisted rotenone-induced changes in cell morphology, loss of cell viability, accumulation of α-synuclein and pS129 α-synuclein, mitochondrial ROS production and mitochondria-mediated apoptosis. The basal and maximal oxygen consumption, mitochondrial coupling efficiency, basal and oligomycin-sensitive ATP and complex I activity in cell model were significantly increased in rotenone + NDI1 group compared to rotenone + vector group. NDI1 was efficiently expressed in dopaminergic neurons in the right SNpc without obvious adverse effects. The rotation number to the right side (NDI1-treated side) was significantly increased compared to that to the left side (untreated side) in mouse model. The number of viable dopaminergic neurons, the expression of tyrosine hydroxylase, total and maximal oxygen consumption, mitochondrial coupling efficiency and complex I enzyme activity in right substantia nigra, and the content of dopamine in right striatum were significantly increased in rotenone + NDI1 group compared to rotenone + vector group.ConclusionYeast NDI1 can rescue the defect of oxidative phosphorylation in rotenone-induced PD cell and mouse models, and ameliorate neurobehavioral and neuropathological damages. The results may provide a basis for the yeast NDI1 gene therapy of sporadic PD caused by mitochondrial complex I dysfunction.

Expert consensus on screening, diagnosis and treatment of multiple carboxylase deficiency.

Multiple carboxylase deficiency (MCD) includes autosomal recessive holocarboxylase synthetase (HLCS) deficiency and biotinidase (BTD) deficiency, which are caused by and gene mutations respectively. Neonatal screening for HLCS deficiency is based on 3-hydroxyisovaleryl carnitine in dry blood filter paper, and BTD deficiency is based on BTD activity determination. HLCS deficiency and BTD deficiency are characterized by neurocutaneous syndrome and organic aciduria, however, they are different in onset age, neurological symptoms and metabolic decompensation, which needed to be differentiated from acquired biotin deficiency or other genetic metabolic diseases. The diagnosis of the disease requires a combination of biochemical characteristics of hematuria, enzyme activity determination and genetic test. Routine biotin doses are effective for most MCD patients. This consensus is intended to benefit early screening and diagnosis of MCD.

First Isolation and Identification of Neopestalotiopsis clavispora Causing Postharvest Rot of Rosa sterilis and Its Control with Methyl Jasmonate and Calcium Chloride

Postharvest rot is a major issue in fruit. However, the cause of postharvest rot on R. sterilis fruit has not been clarified, and there are few studies on the disease control. In this study, the fungus causing postharvest rot is isolated from the symptomatic R. sterilis fruit, and identified by morphological characteristic, pathogenicity test and molecular identification. Moreover, the effects of methyl jasmonate (MeJA) or calcium chloride (CaCl2) alone and their combination on disease resistance to fruit rot were assessed by the determination of defense-related enzyme activity and other indicators. N. clavispora was identified as the main fungus causing the postharvest rot of R. sterilis fruit. The infected fruits were treated with MeJA and CaCl2, and these partially controlled the disease, were additive in effectiveness when used together, increased retention of vitamin C content and fruit firmness, and both enhanced and improved the retention of PAL, POD and PPO activities. The treatment of 500 μL/L MeJA and 3% CaCl2 resulted in the high inhibition of the disease. To our knowledge, this is the first report of N. clavispora causing R. sterilis fruit rot, and the combined treatment is a promising method for controlling postharvest rot on R. sterilis fruit.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

The Influence of Organic and Conventional Cultivation Patterns on Physicochemical Property, Enzyme Activity and Microbial Community Characteristics of Paddy Soil

Cultivation patterns can cause soil structure alteration. However, few studies have clarified the influence of cultivation pattern and soil depth on soil. The purpose of this experiment was to study the community characteristics of soil microorganisms in the 0–10 cm and 10–20 cm layers beneath paddy fields under organic and conventional cultivation patterns, and reveal the response mechanism of microbial community to cultivation patterns through the correlation analysis of soil nutrient content, enzyme activity and microbial dominant phyla, so as to provide a theoretical basis for high-yield rice cultivation from the perspective of microorganisms. In this study, four types of soil organic cultivation topsoil (OF_S), organic cultivation undersoil (OF_X), conventional cultivation topsoil (CF_S) and conventional cultivation undersoil (CF_X) in paddy fields were collected for nutrient and enzyme activity determination, and composition spectrum analysis of soil microbial community diversity was performed using a high-throughput sequencing platform. The results revealed that organic cultivation increased the contents of alkali-hydrolyzable nitrogen, available phosphorus, available potassium and organic substances in both topsoil and undersoil as well as sucrase and urease activity in the undersoil. α diversity indicated that bacterial abundance in both topsoil and undersoil was organic > conventional cultivation; the microbial diversity index in the undersoil under organic cultivation technique was greater than that of conventional cultivation. A Venn diagram revealed that there was considerable difference in species between topsoil and undersoil under organic and conventional cultivation patterns. The composition of the community structure indicated that Proteobacteria, Acidobacteria, Chloroflexi and Bacteroidetes were the dominant phyla of bacterial communities in paddy fields. Ascomycota and Basidiomycota were the dominant phyla of the fungal community. Cluster analysis results indicated that soil depth of both patterns produced apparent clustering effects on microorganisms. Correlation analysis revealed that contents of various soil nutrients and enzyme activities affected the relative abundance of the dominant bacteria and fungi in varying degrees. Alkali-hydrolyzable nitrogen, available potassium and organic matters were significant factors affecting the dominant phyla of soil. The present study demonstrated that compared with conventional cultivation, organic cultivation improved soil physicochemical property, enhanced soil enzyme activity, and altered soil microbial diversity and bacterial abundance. Soil nutrients, enzyme activity and the microbial community of paddy fields interacted with each other and affected the soil structure together.

Effects of Water Temperature on Activities of Metabolic Enzymes of Juvenile Siganus guttatus

Background: Temperature is one of the most important environmental factors affecting the survival, growth and metabolism of fish. The current study was aimed to study the effects of water temperature on the metabolic enzyme activities of juvenile Siganus guttatus. Methods: The juveniles were domesticated at 28±1°C for two weeks and then the temperature was adjusted to the target temperature groups (31°C, 27°C, 23°C and 19°C) by the gradually increasing or decreasing temperature with the change rate of 2°C per day. The experiment lasted for 70 d. At the end of the experiment, the fish were anesthetized and all the livers were dissected on ice plate and stored in the refrigerator at -80°C for the determination of enzyme activity. Result: The activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), hexokinase (HK) and pyruvate kinase (PK), lipoprotein lipase (LPL) and hepatic lipase (HL) tend to be increased with the reduction of temperature. The above enzymes activities in 19°C group were highest. The activity of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and citrate synthase (CS) was lowest in 19°C. These results suggests that 19°C had exceeded the suitable temperature range for juvenile S. guttatus. At low temperature, S. guttatus mainly use fat for energy, but less anaerobic metabolism for energy.

Bellidifolin from Gentianella acuta (Michx.) Hulten protects H9c2 cells from hydrogen peroxide-induced injury via the PI3K-Akt signal pathway

Cardiovascular disease is the most common disease in the world and the first among the causes of human death. Its morbidity and mortality increase annually, but no effective treatment is available. Therefore, new drugs should be developed to treat cardiovascular disease. Gentianella acuta (Michx.) Hulten (G. acuta) is an important Mongolian medicine in China and elicits protective effects on cardiovascular health. In this study, liquid chromatography-mass spectrometry (LC-MS) combined with network pharmacology was used to screen the main active ingredients and confirm that bellidifolin was one of the main components for the treatment of ischemic heart disease. Then, rat myocardial (H9c2) cells injury model induced by hydrogen peroxide (H2O2) in vitro was established to verify the effect of bellidifolin on oxidative stress stimulation, including determination of antioxidant enzyme activity and apoptosis. Transcriptome sequencing, qRT-PCR, and western blot were performed to further verify the antioxidant stress mechanism of bellidifolin. Results showed that bellidifolin pretreatment decreased the rate of apoptosis and the levels of lactate dehydrogenase (LDH), creatine kinase (CK), and alanine aminotransferase (ALT). Conversely, it increased the contents of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in a dose-dependent manner, indicating that bellidifolin caused a protective effect on cardiomyocyte injury. Bellidifolin minimized the H2O2-induced cell injury by activating the PI3K-Akt signal pathway and downregulating glycogen synthase kinase-3β (GSK-3β) and p-Akt1/Akt1. Therefore, this work revealed that G. acuta has a good development prospect as an edible medicinal plant in cardiovascular disease. Its bellidifolin component is a potential therapeutic agent for cardiovascular disease induced by oxidative stress damage.

A guide for the evaluation of in vitro bioaccessibility of carotenoids.

Ongoing efforts to improve the nutritional content of carotenoids in food sources to solve global health problems must also consider bioaccessibility and bioavailability of carotenoids. Carotenoid bioaccessibility encompasses carotenoid release from the food matrix, solubilization in lipid emulsion droplets, and transfer to mixed micelles for further absorption into the intestine. Bioavailability refers to the fraction of carotenoids which enters into circulation. To understand the mechanisms controlling bioaccessibility, in vitro digestion methods have been developed and are a useful approach to overcome some of the disadvantages associated with in vivo protocols. The INFOGEST protocol comprises three phases (oral, gastric and intestinal) to simulate adult human digestion of foods, as an international consensus for the conditions of static in vitro digestion. The last steps in any digestion protocol involve isolation of the target food compounds and subsequent analysis, steps not included in the INFOGEST protocol because they may differ depending on specific physical-chemical characteristics and quantification analysis of the target analyte. In this chapter, we describe the INFOGEST protocol which has been adapted for the analysis of carotenoid bioaccessibility, including determination and calculation of enzyme activities according to INFOGEST guidelines, removal of undigested material, collection of mixed micelles containing carotenoids, extraction, storage and subsequent identification and quantification by liquid chromatography analysis of both free and acylated carotenoids.

Micrococcus luteus: A Potential Keratinolytic Bacteria Isolated from Poultry Waste Dumping Site at Namakkal District, Tamil Nadu, India

: In this study, the experimental soil samples were collected from poultry waste dumping site at Namakkal district, Tamil Nadu, India. From the five soil samples, 15 bacterial colonies were isolated based on Keratinolytic activity with source of agar nutrient medium, nitrogen, carbon and feather meal powder. After the incubation period only 6 isolates showed clear zone of hydrolysis around colony in feather meal agar medium incubated at 37 C for 72 h. The Bacterial species were identified by the morphological and biochemical tests compared with Bergey’s manual of systematic bacteriology IV edition. The obtained bacterial genera from poultry waste dumping sites were Escherichia coli, Enterococcus faecalis, Micrococcus luteus, Klebsiella pneumoniae, Proteus sp., Staphylococcus aureus, Salmonella sp. and Pseudomonas aeruginosa. During the Keratinolytic activity the maximum zone of inhibition was noticed in the isolate No.3, and it was identified as Micrococcus luteus. The identified potential keratinolytic bacterial species culture was optimized with different temperature, pH, nitrogen and carbon sources for bulk production of enzyme. The bulk production of enzyme was precipitated with 20% of ammonium sulphate and it was purified with dialysis bag and subjected to assay procedure for determination of enzyme activity. The antibiotic sensitivity was tested on the isolated bacterial isolates and the highest antibiotic resistance was observed in Staphylococcus aureus (75%) and second most Salmonella (70%) and followed by Klebsiella pneumoniae (60%), Enterococcus faecalis (50%) and Escherichia coli (45%) .

Determination of Some Antioxidant Enzyme Activities in Tomato Seeds Grown in Soils of Different Salinity Classes

Toprak tuzluluğu bitkisel üretimde verim kaybına yol açmakla birlikte doğrudan veya dolaylı olarak tohum verim ve kalitesini de etkilemektedir. Iğdır ovası yazları sıcak ve kurak geçmesi, yıllık ortalama yağış miktarının Türkiye ortalamasının altında olması ve taban suyu yüksekliği nedeniyle yüksek tuzlanma potansiyeli taşımaktadır. Bu durum hali hazırda ovanın bir kısım topraklarının tarım dışı kalmasına yol açmıştır. Bu çalışma ile Iğdır üniversitesi Tarımsal Uygulama ve Araştırma Merkezinde bulunan altı farklı toprak tuzluluğuna sahip alanlarda iki yıl hasat edilmiş ‘Süper domates’ genotiplerinde bazı antioksidan enzim aktiviteleri belirlenmeye çalışılmıştır. Ayrıca çalışma sırasında SOD, KAT ve APX enzim aktiviteleri spektrofotometrik olarak belirlenen tohumlarda doğal poliakrilamit jel elektroforezleri (Doğal PAGE) ayrı ayrı yapılmıştır. Her bir enzim için elektroforez sonrası jellere substrat boyama yapılarak enzimlerin varlığı gösterilmiştir. Elde edilen veriler ışığında tuzlu-alkali topraklarda hasat edilen tohumlarda protein aktivitesi düşük belirlenmişken, en yüksek protein aktivitesi tuzlu topraklardan alınan tohumlarda belirlenmiştir. Toprak tuzluluğunun bitkide oluşturduğu tuz stresi ile tuzlu-alkali alanlarda SOD ve KAT enzim aktiviteleri en yüksek bulunmuşken, normal topraklardan hasat edilen topraklarda SOD ve KAT aktiviteleri daha düşük hesaplanmıştır. APX enzim aktivitelerinde ise normal toprak koşullarında tuzlu-alkali toprak koşullarına doğru aktivitenin arttığı gözlenmiştir. Elde edilen sonuçlar toprak tuzluluğu ve alkaliliğine bağlı olarak bitkide tuz stresinin şiddeti arttıkça hasat edilen tohumlardaki antioksidan enzim aktivitelerinin de arttığını göstermiştir.