Leucine Aminopeptidase Activity Research Articles

Crop straw incorporation mediates the impacts of soil aggregate size on greenhouse gas emissions

Greenhouse gas (GHG) emissions from agricultural soil have been widely discussed to combat the risks of global warming. However, GHG emissions at the soil aggregate scale have yet to be elucidated, particularly in association with straw incorporation. In this study, three different sizes of soil aggregates (1–2, 0.25–1, and < 0.25 mm) were incubated in the laboratory at 25 °C for 58 days with and without rapeseed straw (Brassica napus L.) addition to determine the extent to which aggregate sizes contribute mostly to carbon dioxide (CO2) and nitrous oxide (N2O) emissions upon straw addition. Results showed that cumulative CO2 emission in < 0.25 mm fraction (546 mg C kg−1 soil) was significantly (P < 0.001) lower than that in 0.25–1 mm (810 mg C kg−1 soil) and < 1–2 mm (762 mg C kg−1 soil) fractions in straw-unamended treatments. Straw addition increased cumulative CO2 emissions by 7.2-, 5.87-, and 13.1-fold from 1 to 2, 0.25–1, and < 0.25 mm fractions, respectively, compared with those of the corresponding straw-unamended treatments. Straw addition increased cumulative N2O emissions in each size of aggregates, and cumulative N2O emissions in 1–2 mm fraction ranked the first across the straw-unamended and straw-amended treatments. The activities of β-glucosidase, β-cellobiohydrolase, N-acetyl-β-D-glucosaminidase, and leucine aminopeptidase were enhanced by straw addition in each size of aggregates, and < 0.25 mm fraction exerted the lowest enzyme activities. Structural equation modeling and redundancy analysis confirmed that the interaction between soil physiochemical parameters (nitrate nitrogen and dissolved organic C) and specific enzyme activities was the key driver for regulating CO2 and N2O emissions. These results implied that identifying the straw as a function of aggregate‐scale GHG dynamics could improve the mechanistic understanding of global warming.

Response of soil enzyme activity and bacterial community to copper hydroxide nanofertilizer and its ionic analogue under single versus repeated applications

Nanosized agrochemicals like nanofertilizers are being applied to soils. Adverse impacts of nanofertilizers on soil microflora were reported in past studies, but only considering a single application. Repeated applications are however more likely to occur in agriculture. We investigated effects of single versus repeated applications of a copper hydroxide nanofertilizer formulation (NFF) on soil enzyme activity and bacterial community. One or three applications were performed within 21 days to achieve same final level of Cu in soil (48 mg(Cu)/kg: the recommended dose of NFF). Besides, the active ingredient (i.e., copper hydroxide nanotubes (NT)) and dispersing agent (DA) of NFF, and an ionic fertilizer (i.e., CuSO4) were examined. Fluorescein diacetate hydrolase (FDAse), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and urease (URE) showed negligible changes in the activities between the control and DA treatment. Bacterial community abundance, composition and diversity exhibited similar phenomena. Exposures to copper hydroxide NFF and NT or CuSO4 enhanced the activities of FDAse and NAG, weakened the activity of URE, and showed negligible changes in the LAP activity irrespective of single and repeated applications. Concentrations of NO3−-N and NH4+-N in soil were also affected by the application mode of NFF. More importantly, responses of soil bacterial community to copper hydroxide NFF were highly dependent on its application mode, whereas similar responses were observed in the CuSO4 treatment regardless of single or repeated applications. This study provided new insights into environmental risk of copper hydroxide NFF that were ignored in previous studies using a single exposure.

Effects of fluoranthene on digestive enzymes activity and relative growth rate of larvae of lepidopteran species, Lymantria dispar L. and Euproctis chrysorrhoea L.

Fluoranthene is one of the most abundant polycyclic aromatic hydrocarbon pollutants in the environment and it may accumulate in plant leaves which are the main food source for phytophagous insect species. The aim of this study was to establish the effects of dietary fluoranthene on specific activities of digestive enzymes and expression of their isoforms in the midgut, and the relative growth rates of Lymantria dispar and Euproctis chrysorrhoea larvae. Exposure to fluoranthene led to significantly decreased trypsin activity in the midgut of larvae of both species. Leucine aminopeptidase activity decreased significantly in the midgut of L. dispar larvae exposed to the lower concentration of fluoranthene, but that enzyme activity showed the opposite trend in E. chrysorrhoea larvae. There was no pollutant induced changes in lipase activity in L. dispar, while elevated enzyme activity was recorded in the midgut of E. chrysorrhoea larvae exposed to the lower concentration of fluoranthene. Different patterns of expression of enzyme isoforms were noticed. Relative growth rates of both species significantly decreased in fluoranthene treated larvae. These responses indicate to the significance of relationships between physiological changes and fitness-related traits in L. dispar and E. chrysorrhoea larvae affected by pollutant, and contribute to understanding the mechanisms of their adjustment to stressful conditions.

Root-o-Mat: A novel tool for 2D image processing of root-soil interactions and its application in soil zymography

We developed a software tool enabling user-friendly and standardized pre- and post-processing of images of rooted soil by combining image processing techniques such as image registration, calibration, and segmentation in a graphical user interface. The added benefits of this image processing approach include an improved workflow in soil zymography. For evaluation, we conducted a rhizobox experiment with maize and determined the activity of leucine-aminopeptidase before and after glucose addition based on soil zymography. The temporal and spatial distribution of enzyme activity at the root-soil interface can be visualized by Root-o-Mat which offers 1) standardized image pre-processing, 2) calibration, 3) identification of hotspots of various intensity thresholds, 4) spatial analysis for selected roots, 5) inter-active illustration of enzyme activity profile lines, 6) image viewer, and 7) detection of temporal changes of enzyme activity. Registering images of the same rhizobox taken in successive periods allows further temporal and spatial analysis. We conclude that Root-o-Mat simplifies and firmly anchors image processing and image analyses in soil zymography. The new software can be downloaded for free (www.root-o-mat.de).

The microplastisphere: Biodegradable microplastics addition alters soil microbial community structure and function

Plastics accumulating in the environment, especially microplastics (defined as particles <5 mm), can lead to a range of problems and potential loss of ecosystem services. Polyhydroxyalkanoates (PHAs) are biodegradable plastics used in mulch films, and in packaging material to minimize plastic waste and to reduce soil pollution. Little is known, however, about the effect of microbioplastics on soil-plant interactions, especially soil microbial community structure and functioning in agroecosystems. For the first time, we combined zymography (to localize enzyme activity hotspots) with substrate-induced growth respiration to investigate the effect of PHAs addition on soil microbial community structure, growth, and exoenzyme kinetics in the microplastisphere (i.e. interface between soil and microplastic particles) compared to the rhizosphere and bulk soil. We used a common PHAs biopolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and showed that PHBV was readily used by the microbial community as a source of carbon (C) resulting in an increased specific microbial growth rate and a more active microbial biomass in the microplastisphere in comparison to the bulk soil. Higher β-glucosidase and leucine aminopeptidase activities (0.6–5.0 times higher Vmax) and lower enzyme affinities (1.5–2.0 times higher Km) were also detected in the microplastisphere relative to the rhizosphere. Furthermore, the PHBV addition changed the soil bacterial community at different taxonomical levels and increased the alpha diversity, as well as the relative abundance of Acidobacteria and Verrucomicrobia phyla, compared to the untreated soils. Overall, PHBV addition created soil hotspots where C and nutrient turnover is greatly enhanced, mainly driven by the accelerated microbial biomass and activity. In conclusion, microbioplastics have the potential to alter soil ecological functioning and biogeochemical cycling (e.g., SOM decomposition).

Early Life Intervention Using Probiotic Clostridium butyricum Improves Intestinal Development, Immune Response, and Gut Microbiota in Large Yellow Croaker (Larimichthys crocea) Larvae.

Marine fish larvae are vulnerable during the early life period. The early intervention using probiotics may be a promising method to improve growth of fish larvae. In this study, a 30-day feeding trial was conducted to evaluate the effects of early life intervention using probiotic Clostridium butyricum (CB) on growth performance, intestinal development, immune response and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four isonitrogenous and isolipidic diets were formulated with the supplementation of four different levels of CB (5 × 109 CFU g−1), 0.00% (Control), 0.10% (CB1), 0.20% (CB2), and 0.40% (CB3). Results showed that larvae fed diets with CB had significant higher final length than the control group. Meanwhile, larvae fed the diet with 0.10% CB had significant higher final weight and specific growth rate (SGR) than the control group. However, no significant difference in survival rate was observed among dietary treatments. CB supplementation significantly increased the height of intestinal villus and the length of intestinal enterocyte. Similarly, CB supplementation significantly increased the expression of tight zonula occludens-2 (zo-2) and ornithine decarboxylase (odc) than the control group. Larvae fed the diet with 0.20% CB had significant higher lipase and leucine-aminopeptidase (LAP) activity than the control group. Moreover, CB supplementation significantly improved immune enzyme activities than the control group. Sequencing of bacterial 16S rRNA V4-5 region indicated that dietary CB altered intestinal microbiota profile and decreased intestinal microbial diversities of larvae. CB supplementation could effectively increase the abundance of CB, and decrease the abundance of some potential pathogenic bacteria in larval gut. These results revealed that early life intervention using 0.10–0.20% CB could promote growth of large yellow croaker larvae probably through promoting intestinal development, improving immune enzyme activities and modulating gut microbiota.

White Leghorn Chick의 초기 성장단계에서 소화기관의 발달, 소장의 소화 효소 및 간 조직의 항산화 방어작용

We aimed to investigate the age-dependent development of digestive organs, intestinal enzymes, and hepatic antioxidant defense system in White Leghorn chicks aged 0, 3, 7, 14, and 21 days. Body weight (BW) did not significantly change between days 0 and 7 but significantly increased (<i>P</i>&lt;0.05) after day 7. The relative liver weight (g/100 g of BW) was significantly lower at day 0 than at the other ages but markedly increased at days 3 and 7 (<i>P</i>&lt;0.05). The relative pancreatic weight changed similar to the change in liver weight, with the maximum development at 7 days (<i>P</i>&lt;0.05). The relative intestinal and mucosal tissue weights increased rapidly after hatching (<i>P</i>&lt;0.05), with the maximum growth at 7 days. Furthermore, maltase and sucrase activities were significantly higher at day 3 than at day 0 (<i>P</i>&lt;0.05). Leucine aminopeptidase activity was high at day 0 and remained constant as age increased. Superoxide dismutase and glutathione S-transferase activities in the liver were the lowest at day 0 but significantly increased after 7 days (<i>P</i>&lt;0.05). Glutathione peroxidase activity increased significantly after day 14 compared with that at days 0 and 7 (<i>P</i>&lt;0.05). Lipid peroxidation was not affected by age. In conclusion, the digestive organ weights and hydrolase activity of chicks increased rapidly during the first 3 or 7 days post-hatching. Hepatic antioxidant enzyme activity increased simultaneously with the increase in digestive organ weights, after 7 days.

General Strategy for Bioluminescence Sensing of Peptidase Activity In Vivo Based on Tumor-Targeting Probiotic.

The abnormally expressed peptidases in human tissues are associated with many kinds of cancers. Monitoring of endogenous peptidase activity could allow us for pathophysiology elucidation and early clinical diagnosis. Herein, we developed a general strategy for bioluminescence (BL) sensing of peptidase activity in vivo based on tumor-targeting probiotics. The probiotic that harbored a luciferase-encoding plasmid was used to target and colonize tumor and provide luciferase for BL imaging. The peptide-based probes Lc and GPc were applied to track leucine aminopeptidase (LAP) and dipeptidyl peptidase IV (DPPIV) activity, respectively, by simply adding l-leucine and Gly-Pro dipeptides at the N-terminus of d-cysteine, which were specifically controlled by peptidase cleavage and released free d-cysteine to conduct a subsequent click condensation reaction with 2-cyano-6-hydroxybenzothiazole (HCBT) to produce firefly luciferin in situ, giving rise to a strong BL signal. Neither gene modification of cells of interest nor complicated synthesis was required in this BL system. Encouraged by these advantages, we successfully used our probes to monitor LAP and DPPIV activities in vitro and in vivo, respectively. A good linearity between BL and peptidase was obtained in the concentration range of 2.5-40.0 mU/mL with a limit of detection (LOD) of 1.1 mU/mL (55 ng/mL) for LAP and 2.0-40.0 mU/mL with a LOD of 0.78 mU/mL (1.15 ng/mL) for DPPIV, respectively. Additionally, approximately 5-fold (LAP) and 10-fold (DPPIV) differences in the BL signal before and after treatment with a specific inhibitor were also obtained for in vivo BL imaging. All these results reflected the potential application value of our probes in BL sensing of peptidase activity. We envision that our strategy may be a useful approach for monitoring a wide range of peptidases in tumors, especially in primary tumors.

Root hairs and protein addition to soil promote leucine aminopeptidase activity of Hordeum vulgare L

Protein typically represents the largest input of organic nitrogen (N) into soil. Proteases subsequently make this protein available for use by both plants and microorganisms, however, the factors that regulate protein breakdown in the rhizosphere remain limited. Root exudation of carbon (C) and N into soil promotes microbial growth and thus enzyme production, which is further enhanced by root morphological traits such as root hairs. However, it is not clear how inputs of protein from external sources (e.g. necromass) affect enzyme activity in the rhizosphere. Insight into the interaction between protein addition and root morphology will enhance our knowledge of plant and microbial strategies for promoting N acquisition. Using soil zymography, we investigated the spatial distribution of leucine aminopeptidase activity in the rhizosphere of Hordeum vulgare L. (barley) with and without root hairs subject to localised protein addition. Seedlings of barley were grown for two weeks in rhizoboxes and soluble protein was applied 48 h before analysis of leucine aminopeptidase activity. In situ zymography was used to quantitatively visualise leucine aminopeptidase activity while ex situ sampling was used to determine its enzyme kinetics. In the zymograms, we found that mean and maximal leucine aminopeptidase activity was highest in the barley genotype with root hairs and in the presence of soil protein hotspots. This suggests that microorganisms and plant roots in the rhizosphere of genotypes with root hairs have a greater advantage in accessing protein hotspots in the soil. Leucine aminopeptidase activity did not follow the same trends when analysed by in situ zymography and ex situ sampling methods. Therefore, we recommend the use of in situ zymography to detect the spatial distribution of enzymatic hotspots and rhizosphere extent followed by ex situ sampling for assessing enzyme kinetics in the hotspot areas detected by in situ sampling. However, sampling biases must be considered to ensure enzyme activities are being interpreted as the true rhizosphere.

Rational design of a fluorescent probe for the detection of LAP and its application in drug-induced liver injury

Drug-induced liver injury (DILI) has become a common adverse effect in routine clinical practice, which would further cause the disorder of enzymatic system that respond to multiple pathological progresses. Leucine aminopeptidase (LAP) is regarded as a biomarker in the early course of various liver diseases, in this work, a fluorescent probe NCPL was designed and synthesized for the detecting of LAP. NCPL possessed excellent properties including high selectivity, sensitivity and affinity toward LAP, it could real-time image the LAP activity in living cells and tissues. Additionally, the upregulation of LAP under the APAP-induced liver injury model was also illustrated by NCPL. In conclusion, NCPL as a novel tool could be used for the detection of LAP and monitoring liver function in clinic.

Multi-dimensional imaging of endogenous leucine aminopeptidase via fast response fluorescent read-out probe

Abnormal expression of leucine aminopeptidase (LAP) in living system is associated with various diseases. But so far, there is no detailed imaging analysis of LAP distribution in living cells and organisms with fluorescent probes. In this work, we present a fast-response fluorescent read-out probe (NBD-S-Leu) for imaging LAP in living cancer cells and zebrafish models from multi-dimensional. NBD-S-Leu was constructed by incorporating a dipeptide (cysteinyl-leucine: Cys-Leu) into a commercially available fluorescent 4-chloro-7-nitro-2,1,3- benzoxadiazole (NBD-Cl) scaffold with rational design. Upon exposure to LAP, the amide bond in dipeptides will be cleaved, and subsequently, an intramolecular S→N rearrangement will be occurred, resulting in a dramatic fluorescence light-up and realize the chemoselective detection of LAP. The sensing mechanism was fully supported by TD-DFT calculations and HPLC-HRMS analysis. NBD-S-Leu performs well and features excellent water solubility, fast-response (~4 min) to LAP, low cytotoxicity, as well as good tissue-permeable. Furthermore, NBD-S-Leu has been effectively utilized to image endogenous LAP activity in various types of living cells and zebrafish models. Impressively, with the help of 2.5-dimensional (2.5 D) and 3-dimensional (3 D) confocal imaging, the endogenous LAP distribution in different living cells and zebrafish organs were clearly observed from more than one angle for the first time owing to the high signal-to-noise ratio. Collectively, this fluorescent read-out system does not only provide an alternative method for accurate detection of LAP activity but also can help LAP-related pathological analysis in living organisms and screening inhibitors.

Clay Minerals Change the Toxic Effect of Cadmium on the Activities of Leucine Aminopeptidase

Soil leucine aminopeptidase (LAP) is a hydrolytic enzyme involved in the acquisition of nitrogen by microorganisms. In contaminated soils, LAP activity is affected not only by the type and concentration of heavy metals but also by the form of enzyme. Here, we investigated the degree and mechanism of cadmium (Cd) inhibition of soil LAP and purified LAP. We also examined the effect of montmorillonite and kaolinite on LAP and LAP contaminated with Cd. The results showed that Cd inhibition of LAP activity increased with increasing Cd concentration and that Cd exerted noncompetitive inhibition of LAP. The addition of clay minerals decreases LAP activity and the maximum reaction rate ([Formula: see text]), regardless of the presence of Cd. Montmorillonite decreases the affinity of LAP to the substrate ([Formula: see text]), while kaolinite increases the affinity of LAP to the substrate. The clay mineral-immobilized LAP showed an increase in resistance to Cd contamination compared with the free LAP. The results obtained in this study may aid in understanding the toxic effects of heavy metals on soil enzymes.

Extracellular enzyme activity under subzero hypersaline conditions by model cold-adapted bacteria and Arctic sea-ice microbial communities

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 87:99-111 (2021) - DOI: https://doi.org/10.3354/ame01974 Extracellular enzyme activity of model cold-adapted bacteria and Arctic sea-ice microbial communities under subzero hypersaline conditions G. M. Showalter*, J. W. Deming School of Oceanography and Astrobiology Program, University of Washington, Seattle, WA 98195, USA *Corresponding author: gmshowalter@gmail.com ABSTRACT: Bacterially produced extracellular enzymes (EEs) play an important role in the cycling of organic matter in the marine environment, breaking down large compounds to those small enough to be transported across the cell membrane. EEs may play an especially important role within the brines of sea ice, as freezing concentrates both bacteria and organic materials into brine pockets, leading to higher encounter rates between EEs and their substrates. However, whether EEs are able to perform under the extreme conditions of sea-ice brines, particularly during winter, is unknown. Here, we characterized EE activity (EEA) of leucine aminopeptidase produced by the psychrophilic bacterium Colwellia psychrerythraea strain 34H and the cold-tolerant Psychrobacter strain 7E, under analogue sea-ice conditions using a standard fluorescence-based activity assay. EEs produced by the psychrophile were active at the most extreme conditions tested, i.e. temperature of -8°C and salt concentration of 120 ppt, with activity enhanced if the EEs concerned were produced under subzero hypersaline conditions. EEs produced by the Psychrobacter strain were less cold- and salt-active. When high-latitude Arctic samples of sea-ice brine, under-ice water, and the sea-surface microlayer were analyzed using the same assay after a freeze-thaw cycle, EEA was highest in the sea-ice samples, with activity at -10°C and salinity of 142 ppt. Overall, these results indicate that EEA can contribute to the degradation of organic material in sea ice through winter, likely sustaining microbial communities in brine pores in the process and altering the nature of organic material released at spring melt. KEY WORDS: Arctic marine environment · Carbon cycle · Colwellia psychrerythraea strain 34H · Enzymatic activity · Psychrophiles · Sea ice · Psychrobacter strain 7E Full text in pdf format PreviousNextCite this article as: Showalter GM, Deming JW (2021) Extracellular enzyme activity of model cold-adapted bacteria and Arctic sea-ice microbial communities under subzero hypersaline conditions. Aquat Microb Ecol 87:99-111. https://doi.org/10.3354/ame01974 Export citation Mail this link - Contents Mailing Lists - RSS Facebook - Tweet - linkedIn Cited by Published in AME Vol. 87. Online publication date: August 26, 2021 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2021 Inter-Research.

Dynamics of soil specific enzyme activities and temperature sensitivities during grassland succession after farmland abandonment

The influence of plant secondary succession on soil enzyme activities has been increasingly recognized recently. However, the characteristics of specific enzyme activities and temperature sensitivity (Q10) in the rhizosphere remain elusive. We collected rhizosphere and bulk soil samples from a secondary successional series (0, 7, 12, 17, 22, and 32 years after farmland abandonment, and a natural grassland reference) in a typical semi-arid ecosystems on the Loess Plateau of China. The potential activity of soil enzymes, including β-1,4-glucosidase (BG), β-1,4-N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and alkaline phosphatase (AP), was assayed at 37, 20 and 4 °C and expressed as activity per unit of soil organic carbon (specific enzyme activity). Q10 values were calculated at 20–37 °C and 4–20 °C, respectively. The specific enzyme activities of BG, NAG, LAP and AP in bulk soil increased to a maximum at the 17-year site and then significantly decreased in older grasslands. In the rhizosphere, specific enzyme activities increased and then decreased along the successional gradient with significantly higher value at the 7-year site and the 17-year site. The Q10 values of BG activity in bulk soil and the four enzyme’s activities in rhizosphere soil were changed significantly along the successional gradients. In general, the Q10 values at the two temperature ranges differed in bulk soil, but not in the rhizosphere soil. Along the plant successional gradient, the rhizosphere effects of specific enzyme activities gradually changed to negative values in the oldest sites. Our results indicate that plant secondary succession had different impacts on the enzyme activity and characteristics in the rhizosphere and bulk soil, and highlights the importance of temperature sensitivity and rhizosphere effects of enzyme activity along plant secondary successional gradients.

Use of biochar with immobilized free-living N-fixers as mineral fertilizer

It is known that biochar application as soil fertilizer lead to reduce N loss. At the same time because of its porosity biochar is high quality substrate for immobilization microorganisms. The practice of using free-living N fixing bacteria (FLNFB) in organic farming is widely used to increase the nitrogen content in the soil. In this study immobilisation FLNFB on two types of biochar (made of chicken manure and sewage sludge) was applied to increase soil quality and crop productivity. The effectiveness of immobilization has been proven using SEM method: biofilms of FLNFB on the porous surface of both types of biochars are visible in the SEM images. The effectiveness of application 1% by mass biochar with FLNFB was estimated during 30 days vegetation experiment in greenhouse conditions by estimating the chemical composition of the soil (pH, CEC, OM, Corg, Ntot), by estimating change the functional activity of the soil microbial community (soil respiration activity, leucine aminopeptidase enzyme activity), by estimating change the structure of the microbial community (qPCR were used to analyze nifH and 16S rRNA genes in order to study the total count of bacterial and fungi), by estimating change the plants characteristics (chlorophyll content, germination, root and shoot length, biomass). It was found that application biochar with FLNFB lead to higher content of Ntot (0.11-0.19%) in soil in the end of the experiment as comparing than that for soil samples with mineral fertilizer ( 0.09%) and control soil (0.08%). The application biochar with FLNFB lead to significantly increase nifH-genes in soil. The soil leucine aminopeptidase and respiration activity have a similar trend for samples with and without biochar. The use of biochar as fertilizer led to increase chlorophyll content (13-22%) and barley biomass (10-84%) and decrease the length of the root (3-33%) in comparison with the control soil.

A ratiometric fluorescent probe based on quinoline for monitoring and imaging of Leucine aminopeptidase in liver tumor cells

Leucine aminopeptidase (LAP) is known as an important potential biomarker for liver malignancy and it is urgent to develop an intuitive and effective method to monitor the activity of LAP in liver cancer. Although, numerous LAP fluorescent probes had been developed, it is still a challenge to detect LAP activity in liver cancer. Herein, combained with the DFT, we reported a novel galactose-appended hepatoma-specific ratiometric fluorescent probe (Gal-QL-Leu) based on quinoline group for imaging and tracing LAP in liver tumor cells. Probe Gal-QL-Leu demonstrated a obvious ratiometric characteristics, better selectivity, good biocompatibility and high sensitivity. Moreover, the selective imaging of LAP in HepG2, HCT116, A549 and HeLa cells had been achieved with probe Gal-QL-Leu, demonstrating good application prospect in the detection of LAP activity in liver tumor cells.

Community composition and functional genes explain different ecological roles of heterotrophic bacteria attached to two bloom-forming cyanobacterial genera

Eutrophication leads to frequent outbreaks of cyanobacterial blooms, however, the effect of heterotrophic bacteria attached to cyanobacterial cells is unclear. Field investigations were carried out to gain a deeper understanding of the community composition and functional role of heterotrophic bacteria attached to Dolichospermum and Microcystins cells. The significantly positive relationships between Dolichospermum density and total nitrogen (TN) and between Microcystins density and particle nitrogen (PN) indicated the strong nitrogen (N) demand of these two species. The lack of functional genes that mediate the nitrification process in bacteria attached to both Microcystins and Dolichospermum cells indicated that these two genera preferred ammonium (NH4+-N). Dolichospermum cells obtained more available N through N2 fixation, which was expressed by high nitrogenase gene abundance. Bacteria attached to Microcystins cells showed a higher activity of leucine aminopeptidase and a significantly higher abundance of functional genes that mediate dissimilatory nitrate reduction to ammonium (DNRA) than those attached to Dolichospermum cells. The significantly higher abundance of carbon degradation genes and β-glucosidase activity of bacteria attached to Microcystins cells compared with those of bacteria attached to Dolichospermum cells suggested that abundant organic carbon was bound to Microcystins cells, which is a prerequisite for DNRA. In addition, Microcystins cells exhibited a great advantage in soluble reactive phosphorus (SRP) production through high levels of organic phosphorus (P) hydrolysis associated with high levels of phosphatase genes of attached bacteria. In conclusion, bacteria attached to Microcystins cells performed more important functions on NH4+-N and SRP production through ammonification and DNRA, as well as phosphatase hydrolysis respectively, compared to those attached to Dolichospermum. Thus, algal growth is the result of different variables such as nutrient concentration, their ratio and the microbial ability.

Response of Ecoenzymatic Stoichiometry to Soil Physicochemical Properties after Afforestation on Loess Hilly Region

Afforestation has significantly affected interactions between soil nutrients and microbial enzymes. To understand the detailed variations in extracellular enzyme activity and their link with soil nutrient availability after returning farmland (FL) to forest, soil samples were collected from FL and three afforested lands, Robinia pseudoacacia L. forest (RP), Caragana korshinskii Kom. forest (CK), and abandoned land (AL) in the Loess Hilly Region of northern China. The soil enzymes: β-glucosidase (BG), N-acetylglucosaminidase (NAG), alkaline phosphatase (AP), and leucine aminopeptidase (LAP); their stoichiometric ratios; and soil properties such as soil organic carbon, total nitrogen (TN), total phosphorus (TP), dissolved organic carbon, dissolved organic nitrogen, available phosphorus, soil moisture content (SMC), soil bulk density, and pH were measured. The BG activity showed similar trends as AP activity during afforestation, reaching the highest level in CK and showing a value 205% higher than that in FL. The NAG and LAP activities were not altered by afforestation; only the (NAG + LAP) : AP ratio decreased significantly by 44 to 60%. The BG : AP and (NAG + LAP) : AP ratios were strongly correlated with pH. Compared with other soil physicochemical properties, the soil moisture content greatly affected soil enzyme activity. The most relevant nutrients for the enzymes and stoichiometry were TN and TN : TP. Thus, our results suggest that soil enzyme activity and its stoichiometry are sensitive to changes in soil physical and chemical properties and total soil nutrients content, particularly pH, SMC, TN, and TN : TP.

Purification of Leucine aminopeptidase and its correlation with biochemical parameters in sera of subclinical hypothyroidism and hypothyroidism

The aim of this study purification of Leucine aminopeptidase (LAP) from hypothyroidism patients sera and its relation to thyroid hormones, lipid peroxidation levels of subclinical hypothyroidism and hypothyroidism patients, blood samples were collected from (50) healthy subject and (100) patients, consisted of fifty with hypothyroidism and fifty with subclinical hypothyroidism.. The purification is done by addition of ammonium sulfate, dialysis, anion- exchange chromatography and size-exclusion chromatography. FT3, FT4, TSH, LAP, peroxynitrite, malondialdehyde were determined. Results showed the precipitate and concentrated protein appeared five peaks in ion exchange column, LAP activity located in the first and second protein peak, also, results showed single peak for both first and second peaks after eluted in gel filtration chromatography following steps by using SDS-polyacrylamide gel electrophoresis. From current study, it is concluded hypothyroidism (LAP) have two isoenzymes, also, concluded a highly significant increase in LAP, MDA and peroxynitrite levels in the subclinical hypothyroidism and hypothyroidism patients when compared to the healthy subject, and their correlation with FT3, FT4, TSH in the patient group, when compared by the control group that indicate these parameters could be play an effect role in these diseases.

How “hot” are hotspots: Statistically localizing the high-activity areas on soil and rhizosphere images

The topic of microbial hotspots in soil requires not only visualizing their spatial distribution and biochemical analyses, but also statistical approaches to identify these hotspots and separate them from the surrounding activities (background). We hypothesized that each hotspot type (e.g. enzyme activities in the rhizosphere, root exudation, localization of herbicide accumulation) is a result of local process driven by biotic and/or abiotic factors, and the process rates in the hotspots are much faster than those in the soil background. We further hypothesized that the background and hotspot activities in soil belong to different statistical distributions. Consequently, hotspot determination should be based on statistical separation of activities significantly higher than the background. We analyzed for the statistical distributions of grey values on three groups of published images: 1) 14C images of carbon input by roots into the rhizosphere, 2) 14C glyphosate accumulation in the plant, and 3) zymogram of leucine aminopeptidase activity in rooted soil. The two Gaussian distributions were fit (the first representing the background, the second the hotspots) to the distribution of grey values in the images, the parameters (means and standard deviations, SD) of the fitted distributions were calculated, and the background was removed. Thus, we identified hotspots as areas outside of the Mean+2SD image intensity (corresponding to the upper ~ 2.5% of activity, being over 97.5% of background values) and finally, visualized images of solely hotspot locations. Finally, these results were compared with previously used decisions on hotspot intensity thresholding (i.e. Top-25% and 17 standard thresholding approaches in ImageJ) and discussed the advantages of the Mean+2SD as well as Mean+3SD approaches. These advantages include: i) simple unification of the thresholding approach for several imaging methods with various principles of activity distribution, ii) identification of hotspots with various activity levels, iii) analysis of “time-specific” hotspots in temporal sequences of images. Compared with 17 standard thresholding methods, we concluded that objectively elucidating and separating the hotspots should be based on statistical distribution analysis, e.g. using the Mean+2SD or Mean+3SD approaches. This simple Mean+2SD approach delivered suitable results for three groups of images and so, helps to understand the processes responsible for the highest activities and elucidate hotspots.