A colorimetric method to quantify endo-polygalacturonase activity

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.     

A novel colorimetric method to quantify tannase activity of viable bacteria

A novel colorimetric method to quantify tannase activity of viable tannase-producing bacterial strains was developed through application of a visual reading method that was to detect the activity qualitatively. The novel method was sensitive enough to quantify the marginal tannase activity of strains that could not be otherwise measured by conventional spectrophotometric or colorimetric methods.     

Modeling and dynamics of Wolbachia-infected male releases and mating competition on mosquito control

Despite centuries of continuous efforts, mosquito-borne diseases (MBDs) remain enormous health threat of human life worldwide. Lately, the USA government has approved an innovative technology of releasing Wolbachia-infected male mosquitoes to suppress the wild mosquito population. In this paper we first introduce a stage-structured model for natural mosquitos, then we establish a new model considering the releasing of Wolbachia-infected male mosquitoes and the mating competition between the natural male mosquitoes and infected males on the suppression of natural mosquitoes. Dynamical analysis of the two models, including the existence and local stability of the equilibria and bifurcation analysis, reveals the existence of a forward bifurcation or a backward bifurcation with multiple attractors. Moreover, globally dynamical properties are further explored by using Lyapunov function and theory of monotone operators, respectively. Our findings suggest that infected male augmentation itself cannot always guarantee the success of population eradication, but leads to three possible levels of population suppression, so we define the corresponding suppression rate and estimate the minimum release ratio for population eradication. Furthermore, we study how the release ratio of infected males and natural ones, mating competition, the rate of cytoplasmic incompatibility and the basic offspring number affect the suppression rate of natural mosquitoes. Our results show that the successful eradication relies on assessing the reproductive capacity of natural mosquitoes, a selection of suitable Wolbachia strains and an appropriate release amount of infected males. This study will be helpful for public health authorities in designing proper strategies to control vector mosquitoes and prevent the epidemics of MBDs.

     

RT-PCR method to quantify vascular endothelial growth factor expression.

The vascular endothelial growth factor (VEGF) is implicated in the progression of cancers. Its expression is well correlated with tumor growth and metastases. The availability of a rapid and sensitive method to detect the amounts of VEGF mRNA in biological samples of limited size, very small biopsies, or samples containing relatively few cells could provide an interesting prognostic tool for clinicians. We have developed an RT-PCR method that allows us to detect the VEGF mRNA from as little as 3 micrograms total mRNA. We have also shown that this protocol can be generalized to all cell lines tested. This method constitutes a very potent tool for the analysis of VEGF mRNA expression in different contexts.     

Standardization of formaldehyde-induced fluorescence and its measurement to quantify serotonin emission in pulmonary neuroendocrine cells

Summary We describe a modified and standardized quantitative FIF procedure for producing fluorophores and measuring emission intensity of serotonin-containing neuroepithelial bodies (NEB's) in the rabbit lung. This technique, using epifluorescence, was reproduced without significant differences between control groups. Important considerations for reproducibility were: using the same humidity (80% RH) and reaction time (2 h) during the vapor treatment, sectioning at constant relative humidity, avoiding unnecessary heating (sections should not be stretched over a hot plate) and avoiding exposure of sections to light. Optimal emission readings were obtained with sectioning and mounting at 40–50% RH. Readings were reduced by 25% when the mercury light source was switched from 200 W to 100 W. It was also important to let the instruments warm up long enough to avoid drift during quantitation. Each NEB should be subjected to the same duration of light exposure for alignment (30 s) before measuring fluorescence to avoid differences from photodecomposition.Supported by the College of Agriculture and Life Sciences, University of Wisconsin-Madison and from The Council for Tobacco Research, Inc. USA Numbers 1036 and 1437     

An electrochemical method for measuring metabolic activity and counting cells

An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 10⁵ cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed.     

An electrochemical method for measuring metabolic activity and counting cells

An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 10-5 cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed.     

A sensitive method to quantify the terminal differentiation of cultured epidermal cells

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Y1, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.     

A rapid method to quantify pro-oxidant activity in cultures of wood-decaying white-rot fungi

A new, rapid method for evaluation of lipid peroxidation promoting (pro-oxidant) activity in cultures of wood-decaying fungi was developed. The method is based on measurement of the rate of oxygen consumption in the reaction of linoleic acid peroxidation initiated by fungal culture filtrates. The liquid cultures of the white-rot fungi Bjerkandera adusta and Phanerochaete chrysosporium grown on wheat straw-containing glucose-peptone-corn steep liquor medium possessed significant levels of the pro-oxidant activity. Other white-rot fungi producing manganese peroxidase (MnP) were also found to show the activity. MnP demonstrated a crucial role as the major pro-oxidant agent in the fungal cultures. The total pro-oxidant activity may be considered as net result of the peroxidation by MnP and the inhibition by antioxidant compounds present in the fungal culture fluids.     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.     

Entamoeba histolytica: a simplified method to quantify its cytotoxicity

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.     

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.     

A method to quantify frequency and duration of sustained low-level muscle activity as a risk factor for musculoskeletal discomfort

AimThe purpose of this paper was to describe and evaluate different aspects of muscle activity patterns associated with musculoskeletal discomfort/pain.MethodSurface electromyography (sEMG) of the right upper trapezius and the right extensor digitorum muscles was conducted continuously during one working day in 19 male forest machine operators driving harvesters, 20 driving forwarders and 20 researchers at the Forest Research Institute.Perceived discomfort/pain in the right side of the neck and the right forearm was rated morning, noon and afternoon with Borg’s CR-10 scale. Static, median and peak levels of muscle activity were analyzed and the number and total duration of EMG gaps (muscular rest) were calculated. Sustained low-level muscle activity (SULMA) was defined as continuous muscle activity above 0.5% of the maximal EMG activity quantified into 10 periods of predetermined duration intervals from 1.6 to 5 s up to above 20 min. The number of SULMA periods is presented within each interval and as cumulative periods above the already determined levels. The operators handled control levers seated in a fixed position while the researchers performed mainly PC work and other varied tasks.ResultsA positive correlation was found between discomfort/pain in the right upper trapezius muscle region in the afternoon and cumulative SULMA periods above 10 min duration, and a negative correlation to cumulative SULMA periods also including the short durations. No specified patterns were found for discomfort/pain in the right extensor digitorum or for the other EMG measurements. All EMG measurements distinguished to some extent between the occupational groups, especially between machine operators driving harvesters and researchers.ConclusionsNumber of SULMA periods longer than 10 min per hour was positively correlated, and predominantly short periods were negatively correlated, to complaints in the neck region. This seems promising in order to find duration limits for sustained low-level muscle activity as a risk factor for musculoskeletal disorders.     

A colorimetric method for measuring the esterolytic activity of elastase

This paper describes a new colorimetric method for measuring the activity of elastase. The substrate used is acetyl-l-alanyl-l-alanyl-l-alanine-methyl ester. Its concentration is determined before and after enzymatic action by the hydroxamic acid method of Hestrin. The procedure described is simpler and more rapid than the usual potentiometric method and may be adapted to serial assays. With a 1-hr test, elastase concentrations as low as 0.2 μg/ml may be assessed accurately.     

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.     

Measurement of pH to quantify urease activity.

The rate of change of pH caused by the hydrolysis of urea was measured for urease solutions of 18 different concentrations. Concentrations were converted into an activity, A (measured in IU/cm3), by using a titrimetric method to assay the urease sample. For activities in the range 0.6-38 IU/cm3, A was related to the initial rate of change of pH, (dpH/dt)0, (measured in s-1) by the empirical relationship: A = 549(dpH/dt)0-1423(dpH/dt)2(0). Values of (dpH/dt)0 were sensitive to changes in the urease activity of about 0.6 IU/cm3.