A method for the accurate measurement of opsonic activity in uterine secretions of the mare

This study was undertaken to develop a method for accurately measuring opsonic activity in the uterine secretions of the mare. Ten mares were used in the study. They ranged in ages from 6 to 19 years and were of various genital health status. Undiluted uterine secretions were collected by inserting a tampon into the uterus during estrus; serum samples were collected simultaneously Opsonic activity in the secretions and serum was analyzed in a chemiluminescence assay, in which zymosan particles were opsonized. Opsonic activity was determined as peak chemiluminescence, time to peak chemiluminescence, and total chemiluminescence (area under curve). The peak chemiluminescence was 16 to 17 times higher when uterine secretions were used for opsonization rather than when buffer was used. Compared to the opsonic activity in serum, the peak chemiluminescence was 21% (P相似文献   

Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

A more accurate profile of Achyrocline satureioides hypocholesterolemic activity

The aim of this study was to investigate the effect of the aqueous extract (AE) of Achyrocline satureioides on serum lipid profile, liver oxidative profile and Na(+),K(+)-ATPase activity of rats submitted to a hyperlipidic diet. The animals were divided into four groups: control (C), AE 10% (A(10)), hyperlipidic (H) and hyperlipidic/AE 10% (HA(10)). In serum, we measured the levels of total cholesterol (TC), high-density lipoprotein, very-low-density lipoprotein, low-density lipoprotein (LDL) and triglyceride (TG). In liver homogenates, we measured the thiobarbituric acid reactive substances, the carbonyl proteins, the non-protein thiols (NPSHs) and the activity of superoxide dismutase, catalase (CAT) and Na(+),K(+)-ATPase. We observed a significant increase in the TC and LDL levels in the H group. A. satureioides prevented these effects, decreased the TG levels in the HA(10) group and increased the NPSH levels in the A(10) and HA(10) groups. The H group showed an increase in the carbonyl protein level and a decrease in CAT and Na(+),K(+)-ATPase activities. With the use of this model, results show that increased levels of lipids are related to a redox imbalance in the liver, which is also related to the inhibition of Na(+),K(+)-ATPase activity, and that chronic administration of the AE of A. satureioides is capable of changing this profile.     

Effect of methodology, dilution, and exposure time on the tuberculocidal activity of glutaraldehyde-based disinfectants.

The Association of Official Analytical Chemists (AOAC) test for assessing the tuberculocidal activity of disinfectants has been shown to be variable. A modified AOAC test, which substituted Middlebrook 7H9 broth as the primary subculture medium and used neutralization by dilution, was compared with the standard AOAC method to assess the mycobactericidal activity of three glutaraldehyde-based disinfectants at 20 degrees C and various exposure times. These changes had a marked effect on results, with the modified AOAC test providing more positive penicylinders per 10 replicates in 12 of the 13 comparisons that provided positive results. These differences were observed with both Mycobacterium bovis (ATCC 35743) and a clinical isolate of Mycobacterium tuberculosis. The effects of various exposure times to and dilutions of the glutaraldehyde-based disinfectants were also examined. The minimum exposure time needed to inactivate reliably M. bovis or M. tuberculosis with 2% glutaraldehyde was 20 min at 20 degrees C. Diluting 2% glutaraldehyde caused a significant decline in mycobactericidal activity. Modification of the standard AOAC test to improve its sensitivity in detecting the failure of disinfectants to inactivate mycobacteria is indicated.     

An accurate method for measurement of aldosterone production

A method is described for isolation of the acid-hydrolysable metabolite of aldosterone in sufficient purity to measure accurately the daily production rate. Values obtained with six hospital patients were 84-131mug./day on a daily intake of 100m-equiv. of Na(+) and 227-464mug./day on a daily intake of 10m-equiv. of Na(+). Corresponding values for aldosterone excretion were also recorded, but these are a poor index of production rate since they represent from 1.6 to 9.8% of the total daily output of aldosterone.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

A more accurate method of comparing first-generation maize hybrids with their parents

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A more accurate method of comparing first-generation maize hybrids with their parents

     

A bioluminescence method for direct measurement of phosphodiesterase activity

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25–10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.     

A simple and accurate method for the measurement of the growth of cell monolayers

Summary A procedure has been developed for the accurate, quick, and simple indexing of the growth of insect cell monolayers with a reticule in the eyepiece of an inverted phasecontrast microscope. A magnification of ×500 is desirable for accuracy and ease of counting. Cell nuclei whose circumferences include any of the 25 points in the reticule are enumerated in each microscopic field. Counts of as few as five fields gave an error of 18.8% per flask. Ten fields gave an error of 11.5% and provided a sufficiently accurate comparison for many purposes. Forty counts allowed the measurement of differences between treatments with an error of 5.85%. A large number of treatments can be handled simultaneously, due to the small number of replicates necessary and the small amount of time required per treatment. When the capacity of different batches of lactalbumin hydrolysate to support growth was tested in our insect tissue culture medium, some of them were found to be suitable and others unsuitable. Doubling time of cell populations was measured, after the ratio of the average area of the nuclei at time 0 to that at timeX was used to multiply the number of nuclei counted at timeX. Average nuclear areas were satisfactorily measured by simple measurements of nuclear diameter on an ocular micrometer. The cell nuclei tended to decrease in area when cell monolayers reached confluence or became crowded. The number of replicates required was reduced, because the same flask could be used several times without disturbing the cell monolayer. The method of counting nuclei in a monolayer by means of reticule points and a phase-contrast microscope can also be adapted to the estimation of the absolute number of cells growing on the bottom of a flask. Portion of a thesis by the senior author submitted for the Ph.D. degree to the Graduate College of the University of Illinois. This research was supported in part by grant GB 20915 from the National Science Foundation and United States Public Health Service grant AI 6392 from the National Institutes of Health.     

New method for evaluation of virucidal activity of antiseptics and disinfectants.

Counting culturable viruses adsorbed to cellulose nitrate filters (the VIRADEN method) is proposed as a simple procedure for the evaluation of the virucidal activity of antiseptics and disinfectants. The virucidal activities of two different doses of iodine, chlorine, glutaraldehyde, and chlorhexidine digluconate on poliovirus 1 were tested with a standardized procedure and with the VIRADEN method. The two procedures assayed provided similar results.     

Application of boundary element method for more accurate localization of EEG dipole sources

The proposed modification of boundary element method (BEM) makes it possible to solve direct EEG problem taking into account individual head geometry. This modification decreases computational cost thus enabling practical usage of BEM for localization of EEG dipole sources. The method developed was applied for error estimation of dipole localization using spherical approximation with the aid of BESA software. It was shown that the localization error of BESA for real head geometry could reach 2 cm.     

A new method for measurement of cyclic AMP phosphodiesterase activity.

The extreme sensitivity of chicken muscle fructose 1,6-bisphosphatase to inhibition by 5'-AMP has been utilized to develop a new method for the assay of cAMP phosphodiesterase activity. In this method, the substrate (cAMP) is first incubated with phosphodiesterase and the amount of 5'-AMP formed is then determined by measuring the degree of inhibition of fructose 1'6-bisphosphatase activity. The present method conveniently employs the spectrophotometric technique and is sensitive enough to detect the conversion of 50 pmol of cAMP to 5'-AMP in 1 ml of reaction mixture. This method is considered particularly valuable for those laboratories that are not equipped with facilities for measuring radioactivity.     

An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N-Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.     

A fluorometric method for measurement of oxygen radical-scavenging activity of water-soluble antioxidants

The relative activities of the antioxidants Trolox, ascorbic acid, uric acid, quercetin, and rutin, and the activities of total antioxidants in serum samples were determined using a fluorometric assay based on the dye 6-carboxyfluoroscein (6C-Fl) as a fluorescent indicator; 2,2'-azobis-2-amidinopropane hydrochloride (AAPH) as a peroxyl radical generator; 6-hydroxy-2,5,7, 8-tetramethyl-1-chroman-2-carboxylic acid (Trolox) as a calibrator; and phosphate buffer (pH 7.0) as a solvent. Incubation of 6C-Fl in 0. 075 M phosphate buffer, in the presence of AAPH at 37 degrees C, resulted in loss of its fluorescence signal at 520 nm with excitation at 495 nm. The antioxidants Trolox, ascorbic acid, and uric acid provided protection of the fluorescence of 6C-Fl, and the relative antioxidant activities, determined by the net protection area under curve technique, were found to be 1:0.4:1, respectively. Trolox and ascorbic acid were used to validate this assay. A linear correlation of the net protection value with the concentration of serum, Trolox, ascorbic acid, and uric acid was demonstrated. Quercetin and rutin were shown to have strong antioxidant activities, nearly 10 times those of vitamin C. This assay is simple, reliable, and suitable for automation to handle many samples and requires few microliters of serum samples.     

A simple method for measurement of phosphoramidon-sensitive endothelin converting enzyme activity.

We have developed a rapid and convenient assay for measurement of the action of endothelin (ET) converting enzyme (ECE) using the scintillation proximity assay (SPA) principle. On incubation of [125I]big ET-1 at 37 degrees C for 0.5-6 hr with an enzyme preparation, the reaction was terminated by the addition of an ET-1-specific antibody formulated in a buffer designed to shift the pH to alkaline. The antibody was allowed to come to equilibrium for 1 hr at room temperature and the amount of ET-1 produced, detected in a single step by the addition of protein A SPA beads. Using this assay, ECE activities of enzyme preparations obtained from porcine cultured endothelial cells and rat lung were clearly detected. These activities were inhibited by phosphoramidon in a concentration-dependent manner. The SPA based assay is homogeneous requiring no separation steps and takes a half day to complete. This method is therefore suitable for the high throughput screening of potential ECE inhibitors.