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Modified atmosphere storage containers with gas-permeable membranes are effective in reducing post-harvest losses, mainly at low temperatures but become ineffective in controlling O 2 and CO 2 at temperatures above 3 °C. This study aimed to develop a method to control O 2 in a storage box. This method uses a diffusion tube, that blocks air from entering the box but allows for air exchange when an air blower is activated. The blower ON frequency (BOF) was modelled as a function of temperature, considering the type and amount of produce, blower properties, tube dimension, and the O 2 setpoint. The effects of temperature and gas composition on respiration rate kinetics were analysed with Michaelis-Menten and Arrhenius equations. The model was used to program the microcontroller to control the blower. Validation was performed using a 190 L box containing 25 kg of broccoli. The developed model maintained the desired O 2 concentration under fluctuating temperatures. The BOF values ranged from 25.6 to 549.2 s h−1, for temperature fluctuations between 4 and 23 °C. The time required to reach the 3% O 2 in the box differed depending on the blower's state. With the blower turned ON from the beginning, it took 24.9 h. However, when the blower was initially turned OFF, it took 11.1 h. Despite temperature changes, the system maintained O 2 at 3.8 ± 0.29% and CO 2 at 14.4 ± 0.66%. The system is promising for commercial use and best suited for CO 2 tolerant produce because it requires a separate mathematical model and control mechanism. • Simple method to control gas concentrations under fluctuating temperature. • Real-time O 2 control in storage based on temperature sensor and predictive model. • Mathematical model guides real-time air-blower activation for maintaining O 2. • Suitable for CO 2 -tolerant produce or when combined with CO 2 removal tools. [ABSTRACT FROM AUTHOR]

Desiccation is a severe survival problem for organisms. We have been studying the desiccation tolerance mechanisms in the true slime mold Physarum polycephalum. We measured the trehalose content of P. polycephalum vegetative cells (plasmodia) and drought cells (sclerotia). Surprisingly, we found that the content in sclerotia was about 473-fold greater than in the plasmodia. We then examined trehalose metabolism-related genes via RNAseq, and consequently found that trehalose 6-phosphate phosphorylase (T6pp) expression levels increased following desiccation. Next, we cloned and expressed the genes for T6pp, trehalose 6-phosphate synthase/phosphatase (Tps/Tpp), maltooligosyltrehalose trehalohydrolase (TreZ), and maltooligosyltrehalose synthase (TreY) in E. coli. Incidentally, TreY and TreZ clones have been reported in several prokaryotes, but not in eukaryotes. This report in P. polycephalum is the first evidence of their presence in a eukaryote species. Recombinant T6pp, TreY, and TreZ were purified and confirmed to be active. Our results showed that these enzymes catalyze reactions related to trehalose production, and their reaction kinetics follow the Michaelis-Menten equation. The t6pp mRNA levels of the sclerotia were about 15-fold higher than in the plasmodia. In contrast, the expression levels of TreZ and TreY showed no significant change between the sclerotia and plasmodia. Thus, T6pp is probably related to desiccation tolerance, whereas the contribution of TreY and TreZ is insufficient to account for the considerable accumulation of trehalose in sclerotia. • Study for the desiccation tolerance mechanisms in a true slime mold Physarum polycephalum. • A large amount of trehalose was accumulated in the drought cells (sclerotia). • The genes (t6pp , tps/tpp , treZ , treY) related to trehalose metabolism were cloned. • T6pp is probably related to desiccation tolerance. • This is the first report of treY and treZ genes from eukaryotic species. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bi 2 Fe 4 O 9 nanozyme as a simple and low-cost colorimetric sensor was developed for easy dopamine detection. • The sensor achieved good performance with a linear range from 0.15 to 50 μM and a detection limit of 51 nM. • The nanozyme was reused up to 4 cycles without a significant decrease in its performance. This work describes the preparation of ternary bismuth ferrite oxide nanoparticles (Bi 2 Fe 4 O 9 NPs) with an enzyme mimetic activity for dopamine (DA) qualitative and quantitative detection. Bi 2 Fe 4 O 9 NPs were prepared using a facile, low cost, and one-pot hydrothermal treatment. The chemical composition, morphology, and optical properties of Bi 2 Fe 4 O 9 nanozyme were characterized using different techniques such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction pattern (XRD), X-ray photoelectron spectroscopy (XPS), thermo-gravimetric analysis (TGA), dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM) imaging, FESEM-energy dispersive X-ray spectroscopy (EDS), UV–vis absorption, and fluorescence emission spectroscopy. Bi 2 Fe 4 O 9 NPs were utilized to catalyze the oxidation of a typical chromogenic peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), to form the blue-colored oxidized product (oxTMB), in the presence of hydrogen peroxide (H 2 O 2). All reactions occurred in acetate buffer solution (pH 3.5) to generate hydroxyl radicals (•OH) and the kinetics were followed by UV–vis absorbance at 654 nm. The steady-state kinetic parameters were obtained from the Michaelis-Menten equation and exhibited a good catalytic efficiency of Bi 2 Fe 4 O 9 NPs as enzyme mimetics. Michaelis–Menten constant (K m) values were estimated as 0.07 and 0.73 mM for TMB and H 2 O 2 , respectively. The presented method is efficient, rapid, cost-effective, and sensitive for the colorimetric detection of dopamine with a linear range (LR) from 0.15 to 50 μM and a detection limit (LOD) of 51 nM. The proposed colorimetric sensor was successfully applied for the detection of different concentrations of dopamine in spiked fetal bovine serum (FBS) and horse serum (HS) samples. It is anticipated that Bi 2 Fe 4 O 9 nanozyme holds great potential in biomedical analysis and diagnostic applications of dopamine-related diseases. [ABSTRACT FROM AUTHOR]

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme. [Display omitted] • The dependencies of the activity of lysozyme on various surfactants have common features. • The experimental data are well described by the theoretical curve, assuming three ligand binding sites by lysozyme. • It is possible that the activity of lysozyme can be regulated by natural factors that have suitable hydrophobic radicals. [ABSTRACT FROM AUTHOR]

Partial inhibition occurs when an enzyme-inhibitor complex is capable of producing a product, as opposed to complete inhibition where the enzyme-inhibitor complex is completely inactive and cannot produce a product. While not frequently encountered, partial inhibition is also not a rare phenomenon and can potentially have significant repercussions later on in a research project. It is therefore important to recognize partial inhibition when it occurs. However, it cannot be recognized from the standard velocity versus substrate concentration plots or even from other primary plots such as Lineweaver-Burk plots. Fortunately, partial inhibition is easily identified by using replots which can be generated from the data already gathered to produce the primary plots. Partial inhibition phenomena are also not usually addressed in introductory textbooks and are often given only brief mention in some articles or books dealing with enzyme kinetics. The same types of approaches can be used to study enzyme activation phenomena, which can be considered the flip-side of inhibition. This review presents a graphical approach to identifying partial inhibition in an attempt to provide a comprehensive treatment for those wishing for or needing additional information in carrying out steady-state kinetic inhibition analyses. [ABSTRACT FROM AUTHOR]

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in K M of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment. [ABSTRACT FROM AUTHOR]

Bioluminescence (BL) imaging based on d -luciferin ( d -luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d -luc–luciferase reaction, an approach to increase intracellular levels of d -luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d -luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d -luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d -luc was higher than that in control cells, and the specific d -luc uptake mediated by OAT1 was saturable with a Michaelis constant (K m ) of 0.23 μM. The interaction between OAT1 and d -luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d -luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent K m of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d -luc could be a rate-limiting step in the d -luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d -luc. Our findings suggest that because OAT1 is capable of transporting d -luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. [ABSTRACT FROM AUTHOR]

Microscopic randomness and the small volumes of living cells combine to generate random fluctuations in molecule concentrations called “noise”. Here I investigate the effect of noise on biochemical reactions obeying Michaelis–Menten kinetics, concluding that substrate noise causes these reactions to slow. I derive a general expression for the time evolution of the joint probability density of chemical species in arbitrarily connected networks of non-linear chemical reactions in small volumes. This equation is a generalization of the chemical master equation (CME), a common tool for investigating stochastic chemical kinetics, extended to reaction networks occurring in small volumes, such as living cells. I apply this equation to a generalized Michaelis-Menten reaction in an open system, deriving the following general result: 〈 p 〉 ≤ p ¯ and 〈 s 〉 ≥ s ¯ , where s ¯ and p ¯ denote the deterministic steady-state concentration of reactant and product species, respectively, and 〈 s 〉 and 〈 p 〉 denote the steady-state ensemble average over independent realizations of a stochastic reaction. Under biologically realistic conditions, namely when substrate is degraded or diluted by cell division, 〈 p 〉 ≤ p ¯ . Consequently, noise slows the rate of in vivo Michaelis–Menten reactions. These predictions are validated by extensive stochastic simulations using Gillespie's exact stochastic simulation algorithm. I specify the conditions under which these effects occur and when they vanish, therefore reconciling discrepancies among previous theoretical investigations of stochastic biochemical reactions. Stochastic slowdown of reaction flux caused by molecular noise in living cells may have functional consequences, which the present theory may be used to quantify. [ABSTRACT FROM AUTHOR]

We propose that the time course of an enzyme reaction following the Michaelis-Menten reaction mechanism can be conveniently described by a newly derived algebraic equation, which includes the Lambert Omega function. Following Northrop's ideas [ Anal. Biochem. 321 , 457–461, 1983], the integrated rate equation contains the Michaelis constant ( K M ) and the specificity number ( k S ≡ k cat / K M ) as adjustable parameters, but not the turnover number k cat . A modification of the usual global-fit approach involves a combinatorial treatment of nominal substrate concentrations being treated as fixed or alternately optimized model parameters. The newly proposed method is compared with the standard approach based on the “initial linear region” of the reaction progress curves, followed by nonlinear fit of initial rates to the hyperbolic Michaelis-Menten equation. A representative set of three chelation-enhanced fluorescence EGFR kinase substrates is used for experimental illustration. In one case, both data analysis methods (linear and nonlinear) produced identical results. However, in another test case, the standard method incorrectly reported a finite (50–70 μM) K M value, whereas the more rigorous global nonlinear fit shows that the K M is immeasurably high. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an environmentally prevalent toxicant posing increasing risk to human health worldwide. As compared to the extensive research in Cd tissue accumulation, little was known about the elimination of Cd, particularly its toxic form, Cd ion (Cd 2 + ). In this study, we aimed to examine whether Cd 2 + is a substrate of multidrug and toxin extrusion proteins (MATEs) that are important in renal xenobiotic elimination. HEK-293 cells overexpressing the human MATE1 (HEK-hMATE1), human MATE2-K (HEK-hMATE2-K) and mouse Mate1 (HEK-mMate1) were used to study the cellular transport and toxicity of Cd 2 + . The cells overexpressing MATEs showed a 2–4 fold increase of Cd 2 + uptake that could be blocked by the MATE inhibitor cimetidine. A saturable transport profile was observed with the Michaelis-Menten constant ( K m ) of 130 ± 15.8 μM for HEK-hMATE1; 139 ± 21.3 μM for HEK-hMATE2-K; and 88.7 ± 13.5 μM for HEK-mMate1, respectively. Cd 2 + could inhibit the uptake of metformin, a substrate of MATE transporters, with the half maximal inhibitory concentration (IC 50 ) of 97.5 ± 6.0 μM, 20.2 ± 2.6 μM, and 49.9 ± 6.9 μM in HEK-hMATE1, HEK-hMATE2-K, and HEK-mMate1 cells, respectively. In addition, hMATE1 could transport preloaded Cd 2 + out of the HEK-hMATE1 cells, thus resulting in a significant decrease of Cd 2 + -induced cytotoxicity. The present study has provided the first evidence supporting that MATEs transport Cd 2 + and may function as cellular elimination machinery in Cd intoxication. [ABSTRACT FROM AUTHOR]

Tobacco (Nicotiana tabacum L.) is a potential phytoremediator that can reduce soil cadmium (Cd) contamination. Pot and hydroponic experiments were conducted to investigate the difference in absorption kinetics, translocation patterns, accumulation capacity, and extraction amounts between two leading tobacco cultivars in China. We studied the chemical forms and subcellular distribution of Cd in the plants to understand the diversity of the detoxification mechanism of the cultivars. The concentration-dependent kinetics of Cd accumulation in leaves, stems, roots, and xylem sap for cultivars Zhongyan 100 (ZY100) and K326, fitted well with the Michaelis-Menten equation. K326 exhibited high biomass, Cd tolerance, Cd translocation, and phytoextraction abilities. The acetic acid, sodium chloride, and water-extractable fractions accounted for > 90% of Cd in all ZY100 tissues but only in K326 roots and stems. Moreover, the acetic acid and NaCl fractions were the predominant storage forms, while the water fraction was the transport form. The ethanol fraction also contributed significantly to Cd storage in K326 leaves. As the Cd treatment increased, more NaCl and water fractions were found in K326 leaves, while only NaCl fractions increased in ZY100 leaves. For subcellular distribution, > 93% Cd proportions were primarily stored in both cultivars' soluble or cell wall fraction. The proportion of Cd in the cell wall fraction of ZY100 roots was less than that of K326, while that proportion in the soluble fraction in ZY100 leaves was higher than in K326 leaves. These findings demonstrate that Cd accumulation patterns, detoxification, and storage strategies differ between the cultivars, providing a deeper understanding of Cd tolerance and accumulation mechanism in tobacco plants. It also guides the screening of germplasm resources or gene modification to improve the Cd phytoextraction efficiency of tobacco. [Display omitted] • Kinetics of Cd uptake and xylem translocation in two tobacco cultivars were studied. • K326 has high biomass, Cd tolerance, Cd translocation, and phytoextraction abilities. • Water fraction was the predominant Cd transport chemical form in tobacco. • Cd was primarily stored in vacuole or cell wall as the acetic acid or NaCl fraction. • Ethanol fraction also contributed markedly to Cd storage and transfer in K326 leaves. [ABSTRACT FROM AUTHOR]

In this article, a new approach—namely, the extended Parker–Sochacki method (EPSM)—is presented for solving the Michaelis–Menten nonlinear enzymatic reaction model. The Parker–Sochacki method (PSM) is combined with a new resummation method called the Sumudu–Padé resummation method to obtain approximate analytical solutions for the model. The obtained solutions by the proposed approach are compared with the solutions of PSM and the Runge–Kutta numerical method (RKM). The comparison proves the practicality, efficiency, and correctness of the presented approach. It serves as a basis for solving other nonlinear biochemical reaction models in the future. [ABSTRACT FROM AUTHOR]

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. [ABSTRACT FROM AUTHOR]

[Display omitted] • Investigation of A. elasticus lead to new dihydrobenzoxanthones inhibiting BNA. • Eight compounds, including two new, were characterized by spectroscopic methods. • Kinetic parameters determined by Michaelis-Menten equation double reciprocal plots. • Binding affinities between the BNA and compounds were measured by FS quenching. • The binding site was found by molecular docking experiments. Artocarpus elasticus is a popular fruit tree in the tropical regions. Primary screenings of methanol extracts of the root bark confirmed its potent inhibition of bacterial neuraminidase (BNA), which plays an essential role in the pathogenesis of many microbial diseases. Assessments of the responsible phytochemicals were conducted by isolating eight compounds (1 – 8) and two of them (6 and 8) were identified as new compounds. Among the isolates, the dihydrobenzoxanthones attained the highest BNA inhibition with IC 50 values of 0.5 ∼ 3.9 µM. Further investigation of the inhibitory mechanism by Lineweaver-Burk plots revealed the phytochemicals to function as reversible noncompetitive inhibitors. Fluorescence quenching showed their binding affinities were highly correlated with their inhibitory potential dose-dependently. Molecular docking experiments suggested the dihydrobenzoxanthones (4 and 6) as noncompetitive inhibitors of BNA with unique interaction with Tyr435 of BNA in comparison with the mother flavonoid (7). [ABSTRACT FROM AUTHOR]

We quantify the strength of miRNA–target interactions with MIRZA, a recently introduced biophysical model. We show that computationally predicted energies of interaction correlate strongly with the energies of interaction estimated from biochemical measurements of Michaelis–Menten constants. We further show that the accuracy of the MIRZA model can be improved taking into account recently emerged experimental data types. In particular, we use chimeric miRNA–mRNA sequences to infer a MIRZA–CHIMERA model and we provide a framework for inferring a similar model from measurements of rate constants of miRNA–mRNA interaction in the context of Argonaute proteins. Finally, based on a simple model of miRNA-based regulation, we discuss the importance of interaction energy and its variability between targets for the modulation of miRNA target expression in vivo . [ABSTRACT FROM AUTHOR]

Determination of individual rate constants for enzyme-catalyzed reactions is central to the understanding of their mechanism of action and is commonly obtained by stopped-flow kinetic experiments. However, most natural substrates either do not fluoresce/absorb or lack a significant change in their spectra while reacting and, therefore, are frequently chemically modified to render adequate molecules for their spectroscopic detection. Here, isothermal titration calorimetry (ITC) was used to obtain Michaelis–Menten plots for the trypsin-catalyzed hydrolysis of several substrates at different temperatures (278–318 K): four spectrophotometrically blind lysine and arginine N-free esters, one N-substituted arginine ester, and one amide. A global fitting of these data provided the individual rate constants and activation energies for the acylation and deacylation reactions, and the ratio of the formation and dissociation rates of the enzyme–substrate complex, leading also to the corresponding free energies of activation. The results indicate that for lysine and arginine N-free esters deacylation is the rate-limiting step, but for the N-substituted ester and the amide acylation is the slowest step. It is shown that ITC is able to produce quality kinetic data and is particularly well suited for those enzymatic reactions that cannot be measured by absorption or fluorescence spectroscopy. [ABSTRACT FROM AUTHOR]

Signaling cascades proliferate signals received on the cell membrane to the nucleus. While noise filtering, ultra-sensitive switches, and signal amplification have all been shown to be features of such signaling cascades, it is not understood why cascades typically show three or four layers. Using singular perturbation theory, Michaelis–Menten type equations are derived for open enzymatic systems. Cascading these equations we demonstrate that the output signal as a function of time becomes sigmoidal with the addition of more layers. Furthermore, it is shown that the activation time will speed up to a point, after which more layers become superfluous. It is shown that three layers create a reliable sigmoidal response progress curve from a wide variety of time-dependent signaling inputs arriving at the cell membrane, suggesting the evolutionary benefit of the observed cascades. [ABSTRACT FROM AUTHOR]

Bone is a hard-soft biomaterial built through a self-assembly process under genetic regulatory network (GRN) monitoring. This paper aims to capture the behavior of the bone GRN part that controls mineralization by using a mathematical model. Here, we provide an advanced review of empirical evidence about interactions between gene coding (i) transcription factors and (ii) bone proteins. These interactions are modeled with nonlinear differential equations using Michaelis-Menten and Hill functions. Compared to empirical evidence - coming from osteoblasts culture -, the two best systems (among 12 6 = 2 , 985 , 984 possibilities) use factors of inhibition from the start of the activation of each gene. It reveals negative indirect interactions coming from either negative feedback loops or the recently depicted micro-RNAs. The difference between the two systems also lies in the BSP equation and two ways for activating and reducing its production. Thus, it highlights the critical role of BSP in the bone GRN that acts on bone mineralization. Our study provides the first theoretical evidence of osteoblast self-inhibition after activation of the genetic regulatory network controlling mineralization with this work. [ABSTRACT FROM AUTHOR]

The present work aims to provide insight into interactions between trace metals and higher plants, focusing on nickel uptake and its effects in seagrasses at environmentally relevant concentrations. Total and intracellular nickel accumulation kinetics, nickel effects on structural cell components, oxidative stress marker and cellular viability, and the accumulation kinetics-toxic effects relationship were investigated in leaves of Halophila stipulacea plants incubated in seawater under laboratory conditions containing nickel ions at 0.01–10 mg L−1 for 14 days. Nickel accumulation kinetics in H. stipulacea young and older apical leaves followed a Michaelis-Menten-type equation, allowing the calculation of uptake parameters; uptake rate (V c) and equilibrium concentration (C eq) tended to increase with the increase of nickel concentration in the medium. A dose- and uptake parameter-dependent actin filament (AF) and endoplasmic reticulum (ER) impairment was observed, whereas no effects occurred on microtubules and cell ultrastructure. AF disturbance and ER aggregation were firstly observed in differentiated cells at the lowest concentration on the 12th and 14th day, respectively, while AF disruption in meristematic cells firstly occurred at 0.05 mg L−1; the effects appeared earlier and were more acute at higher concentrations. Increased H 2 O 2 levels were detected, while, at the highest exposures, a significant reduction in epidermal cell viability in older leaves occurred. The lowest total nickel concentrations in young leaves associated with AF disturbance onset at nickel exposure concentrations of 0.01–1 mg L−1 varied between 18.98 and 63.93 μg g−1 dry wt; importantly, they were comparable to nickel concentrations detected in seagrass leaves from various locations. The relationships between exposure concentration, uptake kinetic parameters and toxic effect onset were satisfactorily described by regression models. Our findings suggest that (a) nickel may pose a threat to seagrass meadows, (b) H. stipulacea can be regarded as an efficient biomonitor of nickel, (c) AF and ER impairment in seagrass leaves can be considered as early biomarkers of nickel-induced stress, and (d) the regression models obtained can be used as a tool to evaluate ambient nickel levels and to detect ecotoxicologically significant nickel contamination. The data presented can be utilized in the management and conservation of the coastal environment. Image 1 • Ni uptake kinetics in Halophila stipulacea leaves follow a Michaelis-Menten equation. • Ni at environmentally relevant concentrations can be toxic to seagrasses. • Actin filaments and endoplasmic reticulum can be used as early markers of Ni stress. • Regression models describe the relationships between Ni exposure, uptake and effect. • The seagrass H. stipulacea can be regarded as a valuable biomonitor of Ni pollution. [ABSTRACT FROM AUTHOR]

Snake venom prothrombin activators such as Ecarin are readily assayed by continuous spectrophotometric monitoring of p -nitroaniline production in a one step assay containing prothrombin and a p -nitroanilide peptide substrate for thrombin. The coupled reactions result in accelerating p -nitroaniline (pNA) production over the course of the assay giving non-linear progress curves, from which initial velocities are not readily obtained. Most studies therefore resort to approximate estimates of activity, based on the absorbance reached at an arbitrary time. A simple kinetic analysis of the coupled reactions shows that the early points of such curves should be fitted by second order polynomials, representing the accelerating reaction rate in μmol pNA/min/min. The first derivative of the polynomial then gives the increasing velocity of pNA production in μmol pNA/min over the time course of the assay. We demonstrate here that, with the substrate S2238, these rates can be converted to absolute thrombin concentrations using the Michaelis-Menten equation, substituted with values for kcat and Km. These thrombin concentrations increase linearly over the time course of the assay allowing the activity to be expressed in units, defined as μmol product/min, most commonly used to report enzyme activity. Image 1 • Readily implemented Spectrophotometric Microtitre Plate assay for prothrombin activators. • Continuous monitoring of pNA production by thrombin produced in a coupled reaction assay containing prothrombin and S2238. • Utilizes non-linear Progress Curve Analysis based on a fitted equation derived from the coupled reaction model. • Expresses prothrombin activator activity in μmol of thrombin produced per minute. [ABSTRACT FROM AUTHOR]

Nutrient pollution and algal blooms in coastal waters have long been a major concern, and understanding the response of algae to nutrient dynamics is thus essential. The minimum concentration under which Ulva prolifera will not grow, estimated from a nutrient-alga coupling model developed in this study, was 6.5 μmol L−1 for nitrate and 0.27 μmol L−1 for phosphate. The model was parameterized with cultural experiments to examine the effects of nitrate and phosphate on the growth of Ulva prolifera and the development of green tide. The cultural experiments showed that flux of nitrate absorbed by Ulva prolifera increased significantly with the concentration of nitrate or phosphate, which followed the Michaelis-Menten equation, while the flux of phosphate linearly increased with its concentrations. Nitrate concentrations at >25 μmol L−1 had little influence on the absorption of phosphate, but compensating absorption of phosphate occurred when the nitrate concentration was below 15 μmol L−1. Phosphate concentrations had a remarkable enhancement to the absorption of nitrate when phosphate concentration was <1.4 μmol L−1. The absorption rates of nitrate and phosphate by Ulva prolifera followed the same variation trend as nutrient absorption fluxes. The increase in nitrate absorption rates has a limited impact on the relative growth rate of Ulva prolifera , while the increase in phosphate absorption rates has a marked impact on the relative growth rate. Our results provide new insights into the growth mechanism of Ulva prolifera and the development and evolution of green tides in coastal marine environments. Applications of our model should help governmental agencies in environmental remediation and policy making. [ABSTRACT FROM AUTHOR]