Differences In Enzyme Activity Research Articles

Impact of some phytohormones, metabolic inhibitor and agrochemicals on senescing paddy leaves and its relevance to productivity

The influence of hormones viz. Indole acetic acid (IAA), Gibberellic acid (GA3), Cytokinins viz. Benzyladenine (BA), Benzimidazole (BZI), biocides viz. 2,4-Dichlorophenoxy acetic acid (2,4-D), Diathane M-45, Dimecron and metabolic inhibitors viz. Cycloheximide (CHI) and Actinomycin-D (Act-D) on senescence in detached paddy (Oryza sativa L.) leaves was studied under dark and illuminated conditions. Cytokinins were found to be more effective senescence retardants in light than in dark while auxins were effective in light. Biocides proved to be milder senescence retardants. Effective concentration, a concentration away from the ineffective and toxic range, chosen from IAA, GA3, BA, BZI, 2,4-D, Diathane M-45 and Dimecron were 25,100, 5, 100, 25, 1500 and 0.50 ppm respectively. Application of Act-D (inhibitor of mRNA synthesis) with in 15 min after detachment and cycloheximide (inhibitor of cytoplasmic protein synthesis) from the start of the incubation resulted in suppression of peroxidase activity both under light and dark. Catalase activity exhibited an inverse relation with peroxidase activity under both the experimental conditions. It is recommended that attempts must be made in a more sophisticated way to study the changes in activities of different enzymes suspected to be linked with leaf senescence at a deeper level using all modern techniques available so that the exact nature of enzymes, the isoenzymes pattern and their relation with yield can be understood. Such studies may also help in future in manipulating the projects for the improvement of this economically important cereal crop.

Intracellular actions of inositol messengers and their relationship with the metabolic syndrome

Metabolic alterations occur along with immunological alterations, abnormal placental development and endothelial cell dysfunction in preeclampsia. Typical features of placental alterations are an inadequate/incomplete trophoblast invasion of maternal spiral arteries and acute atherosis. Phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway holds a pivotal role in intracellular metabolic pathways as it regulates endothelial cell survival, migration, tube formation and nitric oxide production. The inositol second messenger system (myo- inositol and D-chiro inositol) is involved in metabolic and cell signaling pathways. Pyruvate dehydrogenase complex (PDC) is at the interface between glycolysis and the citric acid cycle and is crucial to the generation of ATP, acetyl CoA and NADH by mitochondria. We showed that the putative insulin mediator inositol phosphoglycan containing D-chiro inositol (IPG-P) has a sigmoidal inhibitory action on PDK in addition to its known linear stimulation of PDP. Thus, at critical levels of these mediators this sigmoidal/linear model amplifies the switchover from the inactive to active form of PDC, a ‘push- pull’ system that combined with the hormonal control of IPG-P, indicates a powerful regulatory function via inhibition of PDK. The presence of zinc is of interest in relation to our reported effects of zinc ions on PDP and PDH kinases, and in the context of the cardioprotective effects of zinc on vascular ischemia-reperfusion injury. This resetting of the PDC to the active non-phosphorylated state and formation of acetyl-CoA may be enhanced by the effect of zinc on the activation of enolase in the glycolytic pathway and by the regulation of the phosphorylation state of the insulin receptor and thus downstream pathways favouring the provision of pyruvate from glucose. The counter effect of zinc in depressing acetyl-CoA oxidation is obtained via inhibition of key enzymes of the tricarboxylic acid cycle driving acetyl-CoA towards the lipogenic route. Manganese is present in high concentrations in IPG-A type (containing myo-inositol) and coordinates lipid synthesis via manganese activation of different enzymes. Characterization of the intracellular action of inositol messengers in the context of a metabolic disease like preeclampsia helps to understand the underlying biochemical mechanisms that occur in placenta and endothelial cells.

Effect of controlled release formulations of diuron and alachlor herbicides on the biochemical activity of agricultural soils

The use of pesticides in agriculture is essential because it reduces the economic losses caused by pests, improving crop yields. In spite of the growing number of studies concerning the development and application of controlled release formulations (CRFs) of pesticides in agricultural soils, there are no studies about the effects of such formulations on the biochemical properties. In this paper the dissipation of diuron and alachlor in three agricultural soils for 127days, applied either as commercial or CRFs, was determined as well as their concomitant effects on soil biochemical properties. Dehydrogenase, urease, β-glucosidase and phosphatase activities were measured thought the experimental period. The application of alachlor as CRF increases its half-life time in soils, whereas no differences were noticed between diuron formulations due to its slower degradation, which takes longer than its release from the CRF. At the end of the incubation period, the enzymatic activities were the same after the use of diuron either as commercial or CRF, recovering the soil previous status. For alachlor formulations, no differences in enzymatic activities were again observed between both formulations, but their levels in soils were enhanced. Therefore, the use of these CRFs does not adversely affect the soil biochemical properties.

Proteome-wide analysis of lysine acetylation in adult Schistosoma japonicum worm

Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in schistosomes, immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry, was used to comprehensively characterize the lysine-acetylated proteins in this parasite. In total, 1109 acetylated proteins and 2393 acetylation sites in S. japonicum were identified, representing the largest acetylome yet reported in a parasite. In a bioinformatic analysis showed that these acetylated proteins were mainly enriched in the biological process categories of metabolism, gene expression, translation, and transport. The classification according to molecular function revealed that the largest class involved the catalytic activity of different enzymes, including oxidoreductase, transferase, and pyrophosphatase activities. Most of the acetylated proteins in the cellular component category occurred in the cytoplasm, membrane, cytoskeleton, and nucleus. These data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. SignificanceSchistosomiasis is one of the world's most prevalent and neglected tropical parasitic zoonotic diseases, and it causes almost 200,000 deaths annually. To control and eradicate schistosomiasis, effective vaccines are urgently required, and drug targets that are essential for schistosome survival must be identified in fundamental studies of schistosome biology. Posttranslational modifications are complex, fundamental, and important mechanisms that regulate the physiological functions of organisms. Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in S. japonicum, we employ immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry to comprehensively characterize the lysine-acetylated proteins in this parasite. The results of our data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. Meanwhile, identifying the mechanisms and proteins targeted by acetylation may also provide a promising avenue for specific drug design and the development of sophisticated therapeutic strategies.

Assessment of forest soil contamination in Krakow surroundings in relation to the type of stand

The aim of the study was to determine the degree of soil pollution in different forest types around Krakow, using biochemical, chemical and magnetic methods. In assessing the degree of soil contamination, the impact of the forest species composition by comparing the surfaces of broadleaf and coniferous forest stands was taken into account. Enzymatic activity was related to other measures of pollution, i.e., heavy metal concentration, content of polycyclic aromatic hydrocarbons and level of magnetic susceptibility. The study was conducted in southern Poland. Krakow was the central point around which the test surfaces were designated. In soil samples pH, soil texture and organic carbon, nitrogen, base cation contents, dehydrogenase and urease activities were determined. Additionally, heavy metal concentration, polycyclic aromatic hydrocarbons (PAHs) content and magnetic susceptibility were recorded. The obtained results confirm the high diversity of forest soil pollution around Krakow agglomeration. Significant differences in both the content of heavy metals, polycyclic aromatic hydrocarbons and values of the magnetic susceptibility were observed in the soils of forest complexes located in different directions away from Krakow and at various distances from Krakow. The level of contamination is related to the historical and current deposition of industrial and urban dusts from Krakow and Silesia. The type of stand had a considerable influence on the content of polycyclic aromatic hydrocarbons. A higher content of aromatic hydrocarbons was observed in the soils of deciduous stands. A high level of magnetic susceptibility was observed in the soils of coniferous stands. The differences in enzymatic activity between the research plots result from different contents of PAHs and different amount of soil organic matter resulting from the species composition.

Association between GSTT1 and GSTM1 Null Polymorphisms with Pre-Malignant Oral Lesions in North Indian Population

The objective of this investigation is to identify the relationship of GSTT1 and GSTM1 gene polymorphisms with risk of cancer in distinct population in North India with histopathological confirmed oral pre-malignant lesions. A range of parameters such as gender, age, tobacco habit, alcohol consumption and stages of pre-malignant were taken into consideration. Blood samples of 34 histopathological confirmed oral pre-malignant cases, were collected and analyzed for GSTT1 and GSTM1 polymorphisms by PCR method. Thus, the polymorphism of GSTM1 and GSTT1 gene involved may results in the differences in enzymatic activity possibly favoring mechanisms that increase the susceptibility to cancer. Hence, out of 100% pre- malignant population, 72.73% is more susceptible to cancer in case of GSTM1. While in the GSTT1 out of 100% pre- malignant population, 54.55% population is more susceptible to cancer. This study conclude that since the polymorphic genotypes were more in tests samples than the null genotypes, it may be associated with an increased risk of cancer from pre- malignant lesions of oral cancer.

Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers

p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Our in silico structural models of human and Dictyostelium p97 showed that the disease-causing human R93C, R155H, and R155C as well as Dictyostelium R154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for the Dictyostelium R154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containing Dictyostelium protein as a novel p97 interaction partner. We categorized all UBX-domain containing Dictyostelium proteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses of Dictyostelium UBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human and Dictyostelium UBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease.

Neuropeptidase activities in plasma after acute restraint stress. Interaction with cortico-limbic areas.

To evaluate the influence of acute restraint stress (ARS) on plasma enkephalinase and oxytocinase activities. ARS modifies basal activities in cortico-limbic regions of rats and induces changes in the correlations observed between these regions. The interactions between plasma and cortico-limbic activities will be also evaluated. Enkephalinase (AlaAP and LeuAP) and oxytocinase (P-LeuAP) activities were fluorometrically determined in plasma of control and stressed rats using aminoacyl-β-naphthylamides (aaNNap), AlaNNap and LeuNNap as substrates. No differences in enzymatic activities were observed between control and stressed animals in plasma. In contrast, highly significant positive and negative correlations between plasma and cortico-limbic regions were demonstrated in controls. Stress conditions significantly alter the pattern of these correlations. The present results clearly support a connection between plasma and brain involving certain neuropeptidase activities that change under stress conditions.

Liver mitochondrial function in ZDF rats during the early stages of diabetes disease.

The aim of this study was to characterize the early alterations of the liver mitochondrial function in ZDF (fa/fa) rats that develop diabetes compared to that of their lean counterparts ZDF (fa/+). Liver mitochondrial function was examined in 11- and 14-week-old ZDF (fa/fa) and ZDF lean (fa/+) rats. Oxygen consumption, H2O2 release, calcium retention capacity (CRC), membrane potential, membrane fluidity, and fatty acid composition were analyzed. State 3 oxygen consumption with palmitoyl-carnitine increases between 11 and 14weeks of age in lean but not in diabetic animals. This response was not seen with other substrates, suggesting that the use of fatty acids is impaired in diabetic rats. H2O2 release was lower in 14-week-old ZDF (fa/fa) rats as compared to ZDF lean (fa/+). These changes were not associated with differences in enzymatic activities of the respiratory complexes, suggesting regulatory mechanisms independent of their expression levels. Membrane fluidity and composition analyses show only slight effects linked to diabetes progression. The most salient feature was a reduction in CRC in the presence of CsA, an effect reflecting PTP dysregulation. Our data suggest few changes of mitochondrial function in ZDF fa/fa rats. At the age of 11weeks, liver mitochondria have mainly a reduced effect of CsA on CRC.

Catechol-O-Methyltransferase Val158Met Polymorphism and Clinical Response to Antipsychotic Treatment in Schizophrenia and Schizo-Affective Disorder Patients: a Meta-Analysis.

Background:The catechol-O-methyltransferase (COMT) enzyme plays a crucial role in dopamine degradation, and the COMT Val158Met polymorphism (rs4680) is associated with significant differences in enzymatic activity and consequently dopamine concentrations in the prefrontal cortex. Multiple studies have analyzed the COMT Val158Met variant in relation to antipsychotic response. Here, we conducted a meta-analysis examining the relationship between COMT Val158Met and antipsychotic response.Methods:Searches using PubMed, Web of Science, and PsycInfo databases (03/01/2015) yielded 23 studies investigating COMT Val158Met variation and antipsychotic response in schizophrenia and schizo-affective disorder. Responders/nonresponders were defined using each study’s original criteria. If no binary response definition was used, authors were asked to define response according to at least 30% Positive and Negative Syndrome Scale score reduction (or equivalent in other scales). Analysis was conducted under a fixed-effects model.Results:Ten studies met inclusion criteria for the meta-analysis. Five additional antipsychotic-treated samples were analyzed for Val158Met and response and included in the meta-analysis (ntotal=1416). Met/Met individuals were significantly more likely to respond than Val-carriers (P=.039, ORMet/Met=1.37, 95% CI: 1.02–1.85). Met/Met patients also experienced significantly greater improvement in positive symptoms relative to Val-carriers (P=.030, SMD=0.24, 95% CI: 0.024–0.46). Posthoc analyses on patients treated with atypical antipsychotics (n=1207) showed that Met/Met patients were significantly more likely to respond relative to Val-carriers (P=.0098, ORMet/Met=1.54, 95% CI: 1.11–2.14), while no difference was observed for typical-antipsychotic-treated patients (n=155) (P=.65).Conclusions:Our findings suggest that the COMT Val158Met polymorphism is associated with response to antipsychotics in schizophrenia and schizo-affective disorder patients. This effect may be more pronounced for atypical antipsychotics.

Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

Potential effect of increasing the water content in the digestibility of microdiets for fish larvae

This study aimed to find a way for increasing the water content after rehydration of microdiet particles for fish larvae and to assess whether such increase contributes to a better disaggregation and digestion of particles within the gut of gilthead seabream larvae (4–15 days after hatching, dph). Four microdiets were prepared with increasing amounts of carboxymethylcellulose (CMC): 0, 20, 40 and 80 g kg−1. The inclusion of CMC resulted in an effective increase of the hydration capacity of particles. Water content after immersion ranged from 800 g kg−1 in the microdiet without CMC to 900 g kg−1 with 80 g kg−1 CMC. Larvae of different ages were fed during 10 h with these microdiets. The microparticles containing 80 g kg−1 CMC were more degraded in the gut of the younger larvae. No clear differences were observed from 10 dph onwards. Differences in enzymatic activities were not evident except for α-amylase in 6 dph larvae. Gene expression of the enzyme precursors did not show any relation to the water content of microdiets. Overall, the results indicate that increasing the water content up to 900 g kg−1 may enhance the disintegration of food particles within the gut of younger larvae and to a lesser extent affect the response of digestive enzyme activities.

COMPARATIVE ASSESSMENT OF THE LABORATORY SELECTED AND ACTIVE DRIED SACCHAROMYCES CEREVISIAE YEAST CULTURE IN BIOTECHNOLOGY OF THE BRANDY PRODUCTION

<p>Samples from different industrial grape cultivars were collected during the vintage season from the vineyard of the winery (the «Shabo» winery Company, located in the Odesa region, Ukraine). The following industrial cultivars of grapes were selected for the research: Chardonnay, Cabernet Sauvignon, Merlot, Sauvignon, Riesling Rhenish, Aligote, Rkatsiteli, Bastardo, Traminer, Telti Kuruk, Grinosh.</p> <p>The grape cultivars were cultivated on the sandy soils in the district located between the Black Sea and the Dnestrovsky estuary. Grape must derived from different grape cultivars was placed into sterile glass flasks to half of the 450ml flask volume. Each flask was carefully closed with a rubber stopper with an injection needle in it. During the fermentation process, it was necessary to remove carbon dioxide, which was present as a result of active anaerobic fermentation processes in the grape must. At the end of grape must fermentation, pure yeast cultures were isolated using traditional microbiological methods by consistent inoculation of a sample into a Petri dish with a few modifications of nutrient selective agar for yeast isolation and cultivation. Primary yeast isolation was carried out using Inhibitory Mold Agar medium (Becton Dickinson Company, USA).</p> <p>The yeast culture morphological properties were analyzed after the primary yeast culture isolation. Yeasts were identified by polymerase chain reaction (PCR) using universal yeast primers. After yeast culture identification, the next step in yeast cultivation was carried out on Wort Agar medium (Becton Dickinson Company, USA). Each isolated, and identified yeast culture was deposited in the Genebank of Japan, MAFF culture Collection, Tsukuba, Ibaraki, Japan and (NCYC) - Yeast Culture Collection (National Collection of Yeast Cultures, Institute of Food Research, Norwich, United Kingdom). Each yeast culture was tested for technological characteristics such as growth resistance to high temperature (+42°C) and low temperature (+6°C), growth at low pH 2.6–3.0 (acid resistance), growth in the presence of 5, 10, and 15% ethanol (ethanol resistance), and growth in the presence of high concentration potassium bisulfite (bisulfite resistance). Hydrosulfide synthesis (H<sub>2</sub>S gassing production) was studied in addition.</p> <p>Parameters of cellular metabolism in yeast suspension, such as concentration of nitrogen, protein, triglicerides, enzymatic activity and total sugar (which include glucose, fructose, and galactose) were determined. Macro- and micro-element concentrations in fermented grape must, which contained pure yeast culture was determined and included: potassium, sodium, calcium, phosphorus, magnesium, iron, chlorides. In addition to identifying parameters of macro- and micro- element concentration in grape must during and following fermentation based on a principle of photometric analysis, carried out using a biochemical analyser Respons-920 (DiaSys Diagnostic Systems GmbH, Germany).</p> <p>Laboratory selected <em>Saccharomyces cerevisiae </em>wine yeast showed high enzymatic activity with short lag phase. Since of fermentation started on third day concentration of Triglicerides, Protein (total), Potassium and Sodium increased and then level of Protein (total) on the 5th day of fermentation twice decreased. Trigliceride concentration on the 5th day of fermentation continued to increase. Concentration of Iron on the 5th day of fermentation increase in geometrical progression, concentration increase in 4-5 times. Contrary Chloride concentration on the 5th day of fermentation decreased in 3-4 times. Enzymatic activity on 3rd day of fermentation maximal for Lactate Dehydrogenase, Alanine aminotransferase, Aspartate aminotransferase, Phosphatase. Since of 5th day of fermentation Enzymatic activity for Lactate Dehydrogenase, Alanine aminotransferase, Aspartate aminotransferase 3-4 times. Especially level of Phosphatase activity very decreased in 6-7 times. Comparative assessment between our Laboratory selected <em>Saccharomyces cerevisiae</em> yeast culture and Dry active commercial <em>Saccharomyces cerevisiae</em> yeast culture did not showed any difference in enzymatic activity. Both groups showed high enzymatic activity on the third day from the start of fermentation and decreasing on the fifth day since of fermentation started.</p> <p><em> Key words: wine yeast, enzymatic activity, cellular metabolism</em><em>, </em><em>Saccharomyces cerevisiae</em><em>.</em><em></em></p>

Testosterone/dihydrotestosterone ratio as a new biomarker for PCOS

Introduction: Polycystic ovary syndrome is a heterogeneous disease with many different aspects including hyperandrogenism and metabolic disturbances. Clinical phenotypes show different patterns of steroid hormones that have been investigated to some extent. The steroid hormone cascade involves the metabolic activity of different enzymes, e.g. 5alpha-reductase, responsible for the conversion of testosterone (TT) to dihydrotestosterone (DHT). This study intended to determine the 5alpha-reductase activity in PCOS patients by calculating the TT/DHT ratio and to further assess the correlation of this ratio with hormonal, anthropometric and metabolic parameters. Methods: Serum samples of 275 PCOS patients fulfilling Rotterdam criteria and 35 BMI-matched, healthy controls were analysed for testosterone, dihydrotestosterone, dehydroepiandrosterone and androstenedione using liquid chromatography/mass spectrometry. TT/DHT ratio was calculated in order to reflect 5alpha-reductase activity. Result: PCOS patients showed significantly higher levels of total testosterone (p < 0.001), free testosterone (p < 0.001) and free DHT (p < 0.001) compared to controls. TT/DHT ratio was significantly higher in PCOS patients (p < 0.001), indicating a lower 5alpha-reductase activity. No difference was found for total DHT levels (p = 0.072). In PCOS patients alone, TT/DHT ratio was significantly higher in obese patients (p < 0.001) and patients with metabolic syndrome (p < 0.001), pathologic oGTT (p < 0.001) or insulin resistance (p < 0.001). Significant correlations of the TT/DHT ratio with various adverse anthropometric, hormonal, lipid and liver parameters and parameters of glucose metabolism were found. Conclusion: Our data show a higher TT/DHT ratio in serum of PCOS patients, indicating a lower 5alpha-reductase activity and adverse effects on the clinical and metabolic appearance.

Methods of plant root exudates analysis: a review

The aim of this review is to summarise current knowledge on methods being used to determine individual compounds and properties of water-soluble plant root exudates. These compounds include amino acids, organic acids and simple sugars, as well as polysaccharides, proteins and organic substances. Qualitative composition of water-soluble root exudates and exudation rate are commonly measured with the aim of consequent synthetic preparation of plant root exudates to be supplied to soil to create artificial rhizosphere for different experimental purposes. Root exudates collection usually requires consequent filtration or centrifugation to remove solids, root detritus and microbial cell debris, and consequent concentration using an evaporator, lyophilizator or ultrafiltration. Methods used for analysis of total groups of compounds (total proteins and total carbohydrates) and total organic carbon are simple. On the other hand, HPLC or GS/MS are commonly used to analyse individual low molecular weight organic molecules (sugars, organic acids and amino acids) with separation using different columns. Other properties such as pH, conductivity or activity of different enzymes as well as gel electrophoresis of proteins are sometimes assessed. All of these methods are discussed in this work.

Enzymatic and Structural Characterization of rTSγ Provides Insights into the Function of rTSβ

In humans, the gene encoding a reverse thymidylate synthase (rTS) is transcribed in the reverse direction of the gene encoding thymidylate synthase (TS) that is involved in DNA biosynthesis. Three isoforms are found: α, β, and γ, with the transcript of the α-isoform overlapping with that of TS. rTSβ has been of interest since the discovery of its overexpression in methotrexate and 5-fluorouracil resistant cell lines. Despite more than 20 years of study, none of the rTS isoforms have been biochemically or structurally characterized. In this study, we identified rTSγ as an l-fuconate dehydratase and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic activities between rTSβ and rTSγ, enabling more informed proposals for the possible function of rTSβ in chemotherapeutic resistance.

Halophyte Plant Communities Affecting Enzyme Activity and Microbes in Saline Soils of the Yellow River Delta in China

The Yellow River Delta covers a large area of saline soil, which needs to be recovered urgently. As the main local halophytes Suaeda salsa, Phragmites australis, and Tamarix chinensis communities play an important role in the improvement of the soil micro‐environment. Therefore, we investigated the effect of these three salt‐tolerant plant communities on soil enzyme activity in the Yellow River Delta in China. Halophyte plant communities influence the soil micro‐ecosystem in the wetland by exerting effects on the physicochemical properties of the soil, and on enzymes and microbial community. In the soil these enzymes affect the redox potential (catalase) and cycling of carbon (dehydrogenase), nitrogen (protease, urease), and phosphorus (acid phosphatase). We analyzed enzyme activity and the relations between the activity of different enzymes, the physicochemical properties of the soil, and the microbial composition. The activity of five enzymes differed significantly between the plant communities. Soil salinity and microbial community composition all influenced enzyme activity. Catalase, protease and urease activity in the soil of the T. chinensis community was significantly higher than in the other two plant communities. Dehydrogenase and acid phosphatase activity were significantly higher in the soil of the P. australis community than in the other two communities. In the succession of the plant community from herbs to woody plants with different salt tolerance mechanisms, a large aboveground biomass may support the growth of soil enzyme activity and soil microorganism diversity. This study showed that the belowground processes were strongly related with plant community succession in salt marsh ecosystem.

An Improved Single Cell Ultrahigh Throughput Screening Method Based on In Vitro Compartmentalization

High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS) has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >108 clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k cat/K M values approaching the diffusion-limited efficiency of ∼108 M−1s−1.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

Abnormal expression of an ADAR2 alternative splicing variant in gliomas downregulates adenosine-to-inosine RNA editing

BackgroundRNA editing is catalyzed by adenosine deaminases acting on RNA (ADARs). ADAR2 is the main enzyme responsible for recoding editing in humans. Adenosine-to-inosine (A-to-I) editing at the Q/R site is reported to be decreased in gliomas; however, the expression of ADAR2 mRNA was not greatly affected.MethodsWe determined ADAR2 mRNA expression in human glioblastoma cell lines and in normal human glial cells by real-time RT-PCR. We also determined ADAR2 mRNA expression in 44 glioma tissues and normal white matter. After identifying an alternative splicing variant (ASV) of ADAR2 in gliomas, we performed sequencing. We then classified glioblastomas based on the presence (+) or absence (–) of the ASV to determine the correlations between ASV + and malignant features of glioblastomas, such as invasion, peritumoral brain edema, and survival time.ResultsThere were no significant differences in ADAR2 mRNA expression among human glioblastoma cell lines or in gliomas compared with normal white matter (all p > 0.05). The ASV, which contained a 47-nucleotide insertion in the ADAR2 mRNA transcript, was detected in the U251 and BT325 cell lines, and in some glioma tissues. The expression rate of ASV differed among gliomas of different grades. ASV + glioblastomas were more malignant than ASV – glioblastomas.ConclusionsADAR2 is a family of enzymes in which ASVs result in differences in enzymatic activity. The ADAR2 ASV may be correlated with the invasiveness of gliomas. Identification of the mechanistic characterization of ADAR2 ASV may have future potential for individualized molecular targeted-therapy for glioma.