Malachite Green Method Research Articles

Humic Acid-Amended Formulation Improves Shelf-Life of Plant Growth-Promoting Rhizobacteria (PGPR) Under Laboratory Conditions

Plant growth-promoting rhizobacteria (PGPR) is a soil bacterium that positively impacts soil and crops. These microbes invade plant roots, promote plant growth, and improve crop yield production. Bacillus subtilis is a type of PGPR with a short shelf-life due to its structural and cellular components, with a non-producing resistance structure (spores). Therefore, optimum formulations must be developed to prolong the bacterial shelf-life by adding humic acid (HA) as an amendment that could benefit the microbes by providing shelter and carbon sources for bacteria. Thus, a study was undertaken to develop a biofertilizer formulation from locally isolated PGPR, using HA as an amendment. Four doses of HA (0, 0.01, 0.05, and 0.1%) were added to tryptic soy broth (TSB) media and inoculated with B. subtilis (UPMB10), Bacillus tequilensis (UPMRB9) and the combination of both strains. The shelf-life was recorded, and viable cells count and optical density were used to determine the bacterial population and growth trend at monthly intervals and endospores detection using the malachite green staining method. After 12 months of incubation, TSB amended with 0.1% HA recorded the highest bacterial population significantly with inoculation of UPMRB9, followed by mixed strains and UPMB10 at 1.8x107 CFUmL-1, 2.8x107 CFUmL-1and 8.9x106 CFUmL-1, respectively. Results showed that a higher concentration of HA has successfully prolonged the bacterial shelf-life with minimal cell loss. Thus, this study has shown that the optimum concentration of humic acid can extend the bacterial shelf-life and improve the quality of a biofertilizer.

Measurement of Total Phosphorus and Polyphosphate in Chlamydomonas reinhardtii.

Phosphorus is an essential nutrient for plants. Green algae usually store excess P as polyphosphate (polyP) in the vacuoles. PolyP, a linear chain of three to hundreds of phosphate residues linked by phosphoanhydride bonds, is important for cell growth. Based on the previous method of polyP purification with silica gel columns (Werner et al., 2005; Canadell et al., 2016) in yeast cells, we developed a protocol to purify and determine the total P and polyP in Chlamydomonas reinhardtii by a quick, simplified, and quantitative method. We use hydrochloric acid or nitric acid to digest polyP or total P in dried cells and analyze P content using the malachite green colorimetric method. This method may be applied to other microalgae.

A novel malachite green staining approach for evaluating the internal freshness of egg by detecting the quality of egg cuticle

The cuticle is the outermost protective film of eggs, whose quality will affect internal freshness. At present, the undamaged detection of eggs freshness relies on hyperspectral imaging technology, which is relatively complicated. In this study, a novel simple approach for evaluating internal freshness of eggs was further explored by detecting the quality of egg cuticle. Malachite green selected as the alternative dye for quality evaluation of adhesive film, the correlation analysis was carried out by collecting the physical and chemical indexes of eggs with different degrees of freshness. It is found that there was a strong positive correlation between the quality of all parts of the cuticle and internal freshness (p < 0.01), and the Pearson correlation coefficient was over 0.85. The Haugh unit and pH of albumen were most strongly correlated with cuticle quality at the top, with the correlation coefficient of 0.919 and −0.906, respectively. The cuticle quality of eggs with contents got worse than that of eggs without contents, and the holes increased when they were stored for 28 days. In this way, malachite green staining method can be used to identify the freshness of egg cuticle, and the quality of egg cuticle can be used to distinguish eggs with different freshness. This study provided a new direction for the study of egg freshness and assisted to distinguish the quality and safety of eggs nondestructively.

Colorimetric Phosphate Detection Using Organic DFB Laser Based Absorption Spectroscopy.

A novel compact laser absorption spectrometer is developed for colorimetric detection. We demonstrate the realization of the system as well as example measurements of phosphate in water samples based on the malachite green (MG) method. A phosphate concentration range of to (which corresponds to a molar concentration range of to ) is investigated. This photometer demonstrates the ease of integration of organic distributed feedback (DFB) lasers and their miniaturizability, leading the way toward optofluidic on-chip absorption spectrometers. We constructed an optically pumped organic second-order DFB laser on a transparent substrate, including a transparent encapsulation layer, to have access to both emission directions of the surface-emitting laser. Using the two different surface emission directions of the laser resonator allows monitoring of the emitted light intensity without using additional optical elements. Based on these advances, it is possible to miniaturize the measurement setup of a laser absorption spectrometer and to measure analytes, such as phosphate.

Allosteric activators of Ocrl1: a novel therapeutic strategy against Lowe syndrome

Lowe Syndrome (LS) is a lethal genetic disease caused by mutations in the OCRL1 gene that unfortunately leads to the early death of affected children and has no cure. However, this project aims to change such scenario.Although most OCRL1 mutations found in patients impair the enzymatic activity of the encoded lipid phosphatase Ocrl1, about half of these changes do not affect residues involved in binding or modification of the substrate (Fig. 1). In other words, there is a high number of patients expressing Ocrl1 mutated proteins with intact binding/catalytic sites but locked in a conformation unable to process lipid substrates. Therefore, we hypothesized that a substantial subset of Ocrl1 patient mutated proteins could re‐acquire functionality by action of drugs able to stabilize the enzymatically active conformer (allosteric activators). Indeed, Using biochemical and cell biological approaches we identified the FDA‐approved drug and chemical chaperone, 4‐Phenyl Butyric Acid (4‐PBA) as able to restore catalytic activity of and to suppress LS cellular phenotypes caused by two different conformationally‐affected Ocrl1 patient mutants (Fig. 2). In addition, we identified 2 additional FDA‐approved drugs (not acting on Ocrl1 but on downstream pathways) able to ameliorate signaling deficiencies and associated LS phenotypes in cells from patients and in an animal model for LS.This project will produce a novel LS theoretical framework (introducing conformational/misfolding disease component valid for a subgroup of LS patients) and since it will address the lack of therapeutic approaches designed to suppress the causes of LS, has high significance. Importantly, drugs like 4‐PBA are currently used in children to ameliorate other conditions (e.g., urea cycle disorders, cystic fibrosis) and can be readily repurposed to LS (i.e., known safety and low risk of adverse toxicology). Therefore, the translational potential and impact of this project is very high. Further, since our investigations unveiled that LS shares some characteristics with other genetic diseases such as ciliopathies.Support or Funding InformationLowe Syndrome Association, Clinical and Translational Science InstitutePurified GST‐Ocrl11‐542.Bacterial cells wire induced to produce GST‐Ocrl11‐542 WT, D451G and H524R using standard methods. A. Example of purified GST‐Ocrl1 fusion proteins (under different dilutions) as detected by Western blotting using a specific antibody. B–D: The enzymatic activity (measured as phosphate released from PIP2 as function of time) of 3 different concentrations (closed circles&gt;open circles&gt;closed triangles) of purified GST‐Ocrl11‐542 WT (B). D451G (C) and H524R (D) was assayed using the malachite green method. Substrate concentration: 37μM. E. Plot of inital rate (V0) against substrate [PI(4,50P2] concentration for WT Ocrl1 adjusted to a Michaelis ‐Menten equation.Figure 1Phenotypes depending on Ocrl1 phosphates activityA. Cells expressing Ocrl1[text missing] were treated with Vehicle (methanol) or 5mM 4‐PBA for 24h. Note the dispersed pattern of localization of the Ocrl1[text missing] conformationally‐affected variant treated with vehicle as opposed to the tightly perinuclear normal localisation acquired in the presence of 4‐PBA. Scale bars: 10μm. B. Ocrl1D451G characteristic dispersed pattern (Fig. 2) and Golgi apparatus fragmentation was also suppressed by 5mM 4‐PBA treatment. C. Catalytic activity of OCrl1D451G was partially restored by 4‐PBA treatment in a dose‐dependent manner.Figure 2

Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68

The analytical determination of phosphate constitutes a fundamental step for the evaluation of catalytic activities of many enzymes involved in the hydrolysis of phosphate-containing biomolecules. The most sensitive colorimetric methods to quantify phosphate are based on measuring the spectral changes produced by the adsorption of malachite green to 12-molybdophosphoric acid. Malachite green methods are time-consuming because they require the preparation of color reagent on the day of use, due to its low stability. In this work we propose a modification of the malachite green method that overcomes these problems and only requires a one-step, ready-to-use stock solution including perchloric acid and Pluronic F68. The improved reaction mixture allowed the quantification of phosphate with a limit of detection of 0.22 μM, a dynamical range up to 80 μM depending on the optical path-length, and a molar absorption coefficient at 640 nm of 94000 M−1cm−1. Color development reaches a steady level within 30 min and remains constant for at least 2 h. The high stability of the color reagent allows long-term storage for at least 1 year. The optimized procedure is especially useful to measure phosphate-containing biomolecule levels and enzyme activities when low values are critical.

Malachite Green Method for Determining Phosphorus Concentration in Diverse Matrices

ABSTRACTSequential phosphorus (P) fractionation is a procedure often used to assess P dynamics in riparian soils and stream sediments. The most commonly used method to quantify P in fractionation extracts is the molybdate blue-ascorbic acid (MB-AA) colorimetric method. Because sequential P fractionation generates a large number of samples, it would be advantageous to use a single, rapid analytical technique to determine P concentrations. Thus, we investigated the malachite green (MG) colorimetric method for determining P concentrations in the diverse sequential fractionation matrices. For the MG method, a 96-well standard microplate was used, and absorbance was read on a universal microplate reader. We found that the MG and MB-AA methods agreed for determining P concentration in extracts of water, 1 M hydrochloric acid (HCl), and concentrated sulfuric acid (H2SO4). Small differences between the two methods were found for extracts of 0.5 M sodium bicarbonate (NaHCO3) and 0.1 M sodium hydroxide (NaOH), and concentrated HCl, most likely related to differing characteristics of the sediments that were analyzed. Despite this discrepancy for some extracts in the sequential extraction, the microplate system-MG method can be valuable because of its simplicity, sensitivity, and rapidity compared to the MB-AA method.

Hydrolysis of ATP, ADP, and AMP is increased in blood plasma of prostate cancer patients.

Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

Rapid spectrophotometric measurement with a microplate reader for determining phosphorus in NaHCO3 soil extracts

Phosphorus (P) in soil extracted with 0.5 mol L−1 NaHCO3 (pH 8.5) (Olsen-P) is used to evaluate crop P requirement and fertilizer recommendation. A microplate reader with 48- and 96-well plate format was proposed to determine Olsen-P, based on molybdenum blue -ascorbic acid (MA) and malachite green (MG) colorimetric method. The results indicated that the sensitivity of Olsen-P analysis was 2.271 and 3.891 mg L−1 P per absorbance unit and the limit of detection (LOD) was 0.015 and 0.029 mg L−1 for the MA colorimetric measurement with the 48 and 96-well microplate, respectively. The corresponding sensitivity values for the MG colorimetric measurement with the 48 and 96-well microplate were 0.331 and 0.623 mg L−1 P per absorbance unit and the LOD values were 0.002 and 0.007 mg L−1, respectively. Soil Olsen-P concentrations obtained by the microplate reader with the MA and MG colorimetric method were significantly correlated with the conventional spectrometer method with R2 > 0.99 and linear slope near 1.0. The relative standard deviation (RSD) of the MA colorimetric method was 3.0%–23.2% with the 96-well microplate and 0.9%–16.7% with the 48-well microplate. For the MG colorimetric method, the RSD values of the 96 and 48-well configuration were 3.3%–37.6% and 2.3%–17.1%, respectively. The RSD with the 48 -well plate was less than that with the 96-well plate regardless of the MA and MG method. The microplate reader with the MA and MG colorimetric techniques could be used for rapidly measuring P in NaHCO3 soil extracts with high throughput.

Modeling, Synthesis and Characterization of Phospho–Penta–Peptide derived from PKA RII Sub Unit: A Candidate Substrate for CaN Assay

Calcineurin (CaN) plays an important role in many pathological conditions like cancer, metabolic aberrations and neurodegenerative disorders. Till date contemporary methods to assay CaN uses p-nitrophenylphosphate (p-NPP) and specific protein phosphatases 2B inhibitor or use of phospho-peptide substrate derived from protein kinase A (PKA) regulatory sub unit type PKARII. Here we have modeled a series of small phospho peptides derived from PKARII and were docked by Molegro Virtual Docking software to find out the binding free enegy and to determine the affinity with modeled Calcineurin catalytic subunit A (CaN A). Further the candidate phospho–penta–peptide (PPP) derived from PKA regulatory sub unit type II was synthesized in house by employing Fmoc chemistry by solution phase method, purified over HPLC and characterized by mass spectrometry. This PPP was employed to assay CaN activity by Malachite Green method found to be a candidate substrate for CaN.

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl2, HMM hydrolyzed 3PP at a maximum rate of 0.016 s−1 HMM−1, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.

A simple, efficient and economic method for obtaining iodate-rich chili pepper based chitosan edible thin film.

The present study was aimed at designing an iodine supplement in form of edible film made of iodate-coated chitosan (CS-IO3). Inclusion of so obtained films in diet can help in preventing thyroid cancer, leading to improved public health. Chili pepper was coated with iodate thin film (1.5µm). The iodate-rich film is ready-to-eat serving valuable nutrients. Stability studies of CS-IO3 film using water dipping revealed that there was no leaching of iodate ion, due to the strong interactions between cationic amino group of chitosan and iodate ion. The film showed no change in its antioxidant activity. Iodate concentration in the film was determined at 620nm selectively, based on the decolorization of malachite green economic method. Iodate content in fruits coated with 1.5% (w/v) CS-IO3 was 11.5mgg-1 of dry film sample. The iodate-rich samples could be stored without much effect on its freshness, indicating a good shelf life.

Association between activities of SOD, MDA and Na+-K+-ATPase in peripheral blood of patients with acute myocardial infarction and the complication of varying degrees of arrhythmia

ObjectiveTo investigate the changes of ambulatory electrocardiography and peripheral blood SOD, MDA and Na+-K+-ATP enzymes in patients of acute myocardial infarction (AMI) complicated with arrhythmia. MethodsFrom January 2012 to March 2015, 135 cases AMI complicated with arrhythmia in our hospital were divided into 2 groups: 70 cases in the AMI uncomplicated with arrhythmia and 65 cases in the AMI complicated with arrhythmia. 62 cases volunteers accepted physical examination in our hospital in the same period were collected as the control group. 24 hour-electrocardiogram detected by ambulatory electrocardiogram (AECG), SOD and MDA in peripheral blood detected by diagnostic reagent kit and Na+-K+-ATP enzymes in peripheral blood detected by malachite green Kit Method phosphate determination method were collected. ROC curve was used to evaluate the prognostic value of SOD, MDA and Na+-K+-ATP enzymes in AMI patients. ResultsCompared with the control group, the patients had unusual ambulatory electrocardiography had increased (P <0.05), peripheral blood SOD and Na+-K+-ATP enzymes had decreased, peripheral blood MDA had increased in 2 AMI groups (P <0.05). Compared with AMI uncomplicated with arrhythmia group, the patients had unusual ambulatory electrocardiography had increased (P <0.05), peripheral blood SOD and Na+-K+-ATP enzymes had decreased, peripheral blood MDA had increased in AMI complicated with arrhythmia group (P <0.05). Among 135 cases AMI patients, 120 (88.9%) survived and 15 (11.1%) died, of whom 11 cases were AMI complicated with arrhythmia group, 4 cases were AMI uncomplicated with arrhythmia group. Compared with the AMI uncomplicated with arrhythmia group, the dead patients were more in the AMI complicated with arrhythmia group (c2 = 4.287, P = 0.038). Compared with the survival group, the SOD and Na+-K+-ATP enzymes were significantly lower (P <0.05) and MDA significantly higher (P <0.05) in the death group. The area under the ROC curve of SOD, MDA and Na+-K+-ATP enzymes were 0.958, 0.954 and 0.993 respectively, and the cut-off values were 30.66 ng/ml, 576.70 nmol/ml and 57.42 nmol/mgh, respectively. ConclusionAmbulatory electrocardiography has a close relationship with the peripheral blood SOD, MDA and Na+-K+-ATP enzymes in AMI patients complicated with arrhythmia, which might predict AMI condition.

Colorimetric analysis is not sufficient to estimate bioavailable phosphorus in a hypersaline aquatic environment

Colorimetric analysis is commonly used to quantify P availability in aquatic environments. However, it may not be adequate to quantify the bioavailability of dissolved organic P compounds. This study aimed to investigate to what extent the colorimetrically measurable P fractions can be utilized by algae, with a particular emphasis on organic P compounds in a hypersaline aquatic environment. In this study, the growth of the marine alga Dunaliella tertiolecta Butcher was observed in axenic hypersaline cultures prepared with different organic P forms and related to the corresponding levels of colorimetrically measured P and total P. The malachite green method was used to colorimetrically analyze the different P forms and inductively coupled plasma-optical emission spectroscopy was used to quantify the total P. The results showed that only creatine P was colorimetrically detectable, of which 87% of its total concentration was measured. The growth of algae in the culture media with organic P compounds did not reflect the colorimetrically measurable organic P compounds. The results from this study imply that colorimetric analysis may not be sufficient to assess P availability to algae in hypersaline conditions where organic P components are the main source of P.

Ecto-5'-nucleotidase/CD73 contributes to the radiosensitivity of T24 human bladder cancer cell line.

Trimodal therapy is a reasonable bladder-preserving option to radical cystectomy. However, many tumors are radioresistive. In this sense, the identification of new prognostic and predictive biomarkers that allow the selection of patients with better responses to radiation therapy would improve outcomes. With the aim of using ecto-5'-nucleotidase/CD73 as a predictive biomarker, the role of this enzyme in the context of radiotherapy in T24 human bladder cancer cell line was investigated. T24 cell line was exposure to a single dose of radiation (4 Gray) and trypan blue assay (pharmacological assays of viability/cumulative population doubling), flow cytometry (cell cycle/cell death/active caspase-3/ecto-5'-nucleotidase/CD73 protein staining), DAPI staining (nuclear morphometric assay), RT-PCR and real-time PCR, malachite green method (ectonucleotidase enzymatic assay), and HPLC (analysis of AMP metabolism) were carried out. T24 cell line in which ecto-5'-nucleotidase/CD73 has been completely silenced (5'KO) was also used. The exposure of T24 cell line to a single dose (4 Gray) of radiation-induced cell death and triggered a transitory increase in ecto-5'-nucleotidase/CD73 expression, increased ectonucleotidase activity, and led to adenosine and inosine accumulation in the extracellular medium. Pharmacological inhibition or knocking out ecto-5'-nucleotidase/CD73 rescued cells' proliferative capacity, reducing their sensitivity to radiation. Our findings show that the induction of ecto-5'-nucleotidase/CD73 by radiation contributes to the radiosensitivity of T24 cell line.

Alpha-bisabolol Promotes Glioma Cell Death by Modulating the Adenosinergic System.

Glioblastoma multiforme is the most malignant type of glioma. Alpha-bisabolol is an essential oil reported as a potent cell death agent. In the present work, we evaluated the effect of alpha-bisabolol on ecto-5'-nucleotidase/CD73, the most well-characterized enzymatic source of adenosine, present in lipid rafts. Glioma cells were treated with alpha-bisabolol and, in some experiments, pre-treated with an A3 antagonist. MTT assay (viability), malachite green method (ecto-5'-nucleotidase/CD73 activity) and quantitative polymerase chain reaction (qPCR) (A3 mRNA) were carried out. Alpha-bisabolol led to a decrease in C6 and U138-MG glioma cells viability, accompanied by an increase in ecto-5'-NT/CD73 activity. Pre-treatment with an A3 antagonist reverted the effect of α-bisabolol with an increase of mRNA expression of this receptor. Our data indicated the participation of ecto-5'-nucleotidase/CD73 and A3 receptor in the anti-proliferative effect of α-bisabolol on glioma cells.

Phosphate-Containing Prescription Medications Contribute to the Daily Phosphate Intake in a Third of Hemodialysis Patients

Hyperphosphatemia is associated with all-cause mortality in hemodialysis (HD) patients and is managed by restricting dietary phosphate. Many patients are unable to adhere to the recommended dietary phosphate limit. We sought to quantify the additional phosphate burden from prescription medication in a hemodialysis population. Cross-sectional study. Adult patients on hemodialysis at a single center. The Health Canada Drug Product Database was used to identify formulations of medications prescribed in an HD program that contain phosphate salts. The manufacturers of formulations containing a phosphate salt were contacted to request the phosphate content per tablet, and amounts were confirmed in select medications by the malachite green method. Prescription bottles of 101 HD patients were evaluated. Reported phosphate contents were used to determine patients' daily phosphate load from prescribed medications. A total 1,744 drug formulations of 124 different medications were reviewed. A total of 185 (11%) contained a phosphate salt. Central nervous system (CNS) and cardiovascular (CVS) medications accounted for 65% and 24% of phosphate-containing medications, respectively. Of HD patients, 30% were taking at least one medication that contained phosphate. The median phosphate burden from prescribed medications was 111 (67-168) mg per day. Knowledge about the phosphate content of commonly prescribed drugs within different classes should influence prescribing patterns. Particular consideration of which formulation of CVS and CNS drugs contain phosphate should be applied when prescribing. Phosphate-containing medications can meaningfully contribute to the daily phosphate load in HD patients; however, this burden will differ based on local dispensing patterns.

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method

The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors.

An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L−1 and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro.

Discovery of a latent calcineurin inhibitory peptide from its autoinhibitory domain by docking, dynamic simulation, and in vitro methods

Autoinhibitory domain (AID) of calcineurin (CN) was discovered two decades ago. Fewer investigations are reported to find out shortest possible peptide from the AID for CN inhibition. Hence, this study has focused on screening of nearly 150 peptide fragments derived from the AID using in silico method. Therefore, we have employed docking studies, aiming to analyze the best pose of AID-derived peptides on CN active site. We also analyzed binding free energy (ΔG) of docked complex using molecular mechanics/generalized Born surface area (MM/GBSA). MM/GBSA predicts two short peptides P1 and P2 found to be lowest binding free energy. Two peptides exhibit better binding affinity with CN, suggests that the possible candidates for potential CN inhibition. Further, the stability of the docked complex was analyzed using molecular dynamic (MD) simulation. MD study shows that CNA:P2 is the most stable complex than CN A:P1 and CN A:AID. Besides, we have synthesized and purified P1 and P2 peptides over high performance liquid chromatography (HPLC) found to be 90.31% and 98.93% of purity, respectively. In addition, AID peptides were characterized over mass spectral analysis. Peptides were subjected to CN inhibitory assay using malachite green method. Where, P1 and P2 exhibit CN inhibition better than AID. In particular, shortest peptide P2 shows highest inhibitory activity than AID. Enzyme assay reveals CN inhibitory activity of P2 peptide is consistent within silico results. In silico and in vitro, results corroborated each other to confirm short peptide P2 can be used as a potential CN inhibitor.