Quality-control method for the determination of biological activity of engineered calcineurin subunit B

The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.     

A radiometric microassay for choline acetyltransferase. Some observations on the spinal cord of the chicken embryo

This paper describes cation-exchange methods for separating acetyl[³H] coenzyme A from [acetyl-³H]choline. Blanks for the routine method were approximately 0.05% of the substrate radioactivity; product recoveries were approximately 97%. The cation-exchange method was moreefficient than the standard methods using either anion-exchange chromatography or periodide precipitation. The cation-exchange method was also morespecific than either of the other two standard methods for estimating choline acetyltransferase (ChAT) activity. ChAT activity was detected in the chicken lumbar spinal cord on embryonic day (E) 2 1/4 with the cation-exchange method. This developmental stage is about 6 hours before the final mitosis of any neuroblast in the ventral horn. Total ChAT activity per lumbar spinal cord increased more than 10,000-fold between E 3 and E 18. Changes in ChAT activity in the lumbar spinal cord following limb-bud extirpation appeared to mirror (with a phase lag) the changes in the number of motoneurons in the lateral motor column.     

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.     

Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia

Tea tree oil, or the essential oil of Melaleuca alternifolia , is becoming increasingly popular as a naturally occurring antimicrobial agent. The antimicrobial activity of eight components of tea tree oil was evaluated using disc diffusion and broth microdilution methods. Attempts were also made to overcome methodological problems encountered with testing compounds which have limited solubility in aqueous media. After assessing media with and without solubilizing agents, the disc diffusion method was used to determine the susceptibility of a range of micro-organisms to 1,8-cineole, 1-terpinen-4-ol, ρ-cymene, linalool, α-terpinene, γ-terpinene, α-terpineol and terpinolene. While the disc diffusion method lacked reproducibility, it was considered useful as a procedure for screening for antimicrobial activity. Terpinen-4-ol was active against all the test organisms while ρ-cymene demonstrated no antimicrobial activity. Linalool and α-terpineol were active against all organisms with the exception of Pseudomonas aeruginosa. Minimum inhibitory and minimum cidal concentrations of each component against Candida albicans, Escherichia coli and Staphylococcus aureus were determined using a broth microdilution method. Modifications to this method overcame solubility and turbidity problems associated with the oil components and allowed the antimicrobial activity of each of the components to be quantified reproducibly. There was reasonable agreement between minimum inhibitory concentrations and zones of inhibition. These results may have significant implications for the future development of tea tree oil as an antimicrobial agent.     

Methods for the measurement of a bacterial enzyme activity in cell lysates and extracts

The kinetic characteristics and regulation of aspartate carbamoyltransferase activity were studied in lysates and cell extracts of Helicobacter pylori by three different methods. Nuclear magnetic resonance spectroscopy, radioactive tracer analysis, and spectrophotometry were employed in conjunction to identify the properties of the enzyme activity and to validate the results obtained with each assay. NMR spectroscopy was the most direct method to provide proof of ACTase activity; radioactive tracer analysis was the most sensitive technique and a microtitre-based colorimetric assay was the most cost-and time-efficient for large scale analyses. Freeze-thawing was adopted as the preferred method for cell lysis in studying enzyme activity in situ. This study showed the benefits of employing several different complementary methods to investigate bacterial enzyme activity.     

Leu-Enkephalin Generally Labeled with Tritium in Studying the Selank Inhibiting Effect on the Enkephalin-Degrading Enzymes of Human Blood Plasma

A method of analysis of enkephalinase activity in blood plasma based on the application of Leu-enkephalin generally labeled with tritium at all its amino acid residues was developed. The method allows the simultaneous estimation of activity of several peptidases in microquantities of tissues. [G-³H]Leu-enkephalin was prepared by the method of solid phase catalytic isotope exchange (120 Ci/mmol) and subjected to proteolysis by the treatment with blood plasma. The resulting radioactive metabolites were separated by HPLC in the presence of the mixture of unlabeled fragments of Leu-enkephalin as internal standards. It was shown that aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases respond for approximately 80%, 2%, and 10% of the total enzymatic activity, respectively. The new pathway of degradation of Leu-enkephalin by carboxypeptidase that provides for 6% of the total enkephalin-degrading activity was discovered. Bestatin was shown to predominantly inhibit aminopeptidases and carboxypeptidases, whereas selank is more specific for carboxypeptidases and dicarboxypeptidases.     

Some effects of turbulence on the activity of the sea urchin Centrostephanus coronatus Verrill

Effects of turbulence on activity of the sea urchin Centrostephanus coronatus Verrill were studied using a new method referred to as the trip-thread method which recorded incidences of emergence from burrows during activity periods. During calm conditions, the number of C. coronatus emerging at 5 m depth was not significantly different from the number emerging at 15 m depth, representing 97% and 92% activity in the populations, respectively; however, during turbulent conditions, the number of individuals emerging at 5 m was significantly less than the number emerging at 15 m, representing 48% and 92% activity, respectively. These results suggest an inverse relationship between turbulence and activity. Although behavioral adaptations, such as reduced activity, allow urchins to occupy turbulent areas, extreme conditions may exclude them entirely. Differences in activity may affect grazing by sea urchins which can significantly affect the species composition and abundance of benthic communities.     

An improved assay method for a pseudomurein-degrading enzyme of Methanobacterium wolfei and the Protoplast formation of Methanobacterium thermoautotrophicum by the enzyme

An improved assay method of a pseudomurein-degrading enzyme and its properties are described. The pseudomurein-degrading enzyme purified from Methanobacterium wolfei autolysate under an anoxic condition was assayed with the cell wall of Methanobacterium thermoautotrophicum as a substrate. By this improved method the enzyme activity was measured quantitatively and reproducibly. Moreover, the cell wall substrate can be stored in a freezer and used as needed, and the time required for an assay was as short as 1 h. The optimum pH and temperature of the enzyme was pH 6.8-7.4 and 75°C, respectively. Although the enzyme lost 50% of the activity upon heating at 75°C for 10 min in the absence of the cell wall substrate, it was more stable against heat inactivation in the presence of the substrate. Furthermore the inactivated enzyme recovered some of the activity by incubating with the substrate. Although the enzyme lost most of the activity under aerobic conditions, the activity was recovered under reducing conditions with Na₂S·9H₂O or DTT (dithiothreitol). The enzyme was also purified under aerobic conditions retaining the same specific activity as the anoxically purified enzyme. Using the partially purified enzyme the conditions preparing protoplasts of M. thermoautotrophicum was established.     

A fluorimetric method for measuring the activity of soil enzymes

Summary A fluorimetric method is described for the measurement of the activity of a range of soil enzymes. The method is based on the measurement of 4-methylumbelliferone (MUB), a fluorescent product liberated on hydrolysis of the enzyme substrate. The main advantage of the method over colorimetric techniques is that separation of MUB from the soil is unnecessary and the method is therefore suitable for routine, automated analyses. The method was used to measure the activity of β-cellobiase, β-galactosaminidase, β-glucosidase and β-xylosidase over a wide range of substrate concentration and in a range of soils. Kinetic parameters are reported for these enzymes. The method was also shown to be suitable for the assay of arylsulphatase and acid and alkaline phosphatase in soil. The technique should be applicable to a wide range of soil hydrolases, using the same assay methods.     

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

An attempt to predict the start,peak and end of a pollen season

The different phases, start, peak and end of the pollen season were predicted using a cumulated activity method. This procedure is a development of the temperature sum method. The temperature was transformed to activity (A):

A = 2**[(T+g)/(10*h)] - k.

T = daily mean temperature over the treshold.

g,h,k = coefficients.

Other weather parameters are also to be included in the future.

The activity is cumulated day by day and the sum is used as an indicator of a specific phase. A cumulated activity model is defined by the above formula with specified values of the three coefficients g, h and k. Four conditions stating criteria for valid cumulation days and the cumulated target sum for the phase in question are to be defined. The model was based on pollen and weather data in Stockholm 1973–1988. The cumulated activity method was found to be best applicable to Ulmus, Betula and Pinus, but less useful for Alnus, Corylus and Quercus. The model was not satisfactory for Poaceae and Artemisia.     

Non-destructive measurement of acid phosphatase activity in the rhizosphere using nitrocellulose membranes and image analysis

We developed a method using nitrocellulose membranes and image analysis to localise and quantify acid phosphatase activity in the rhizosphere of two plant species, one with cluster roots (Dryandra sessilis (Knight) Domin) and another with ectomycorrhizal roots (Pinus taeda L.). Membranes were placed in contact with roots and then treated with a solution of x, α-naphthyl phosphate and Fast Red TR. Acid phosphatase activity was visualised as a red imprint on the membrane. We quantified acid phosphatase activity by image analysis of scanned imprints. The method was used to estimate the spatial distribution of acid phosphatase activity within particular root classes (lateral roots, mycorrhizal roots, root clusters). Over 95% of the acid phosphatase activity of the root system of D. sessilis was associated with cluster roots, and between 20 and 32% of the root surface active. About 26 % of the acid phosphatase activity of the root system of P. taeda was associated with mycorrhizal roots and unsuberised white root tips and less than 10% of the root surface was active, irrespective of root type. This non-destructive method can be used for rapid, semi-quantitative assessment of acid phosphatase activity in the laboratory and in situ. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improvements in the determination of manganese peroxidase activity

The existing method of determining the activity of manganese peroxidase (MnP), produced by Phanerochaete chrysosporium, was improved. 2,6-Dimethoxyphenol at 80 mM was used as a substrate and, after the decolorization of the reaction mixture, H₂O₂ was added and the initial reaction rate was used to determine MnP activity.     

Nonstationary power spectrum analysis of the photic alpha blocking

The dynamics of the EEG activity under light stimulation was investigated in terms of the nonstationary power spectrum method. The alpha wave was blocked by light stimulation, however the blocking was temporary, and the alpha activity recovered to that of the resting state even though under light. Moreover the alpha rhythm was also blocked by light off. The frequencies of the preand post-blocked activity were different. The frequency of the post-blocked wave shifted gradually downward and became stable after 3–4 sec.     

Occurrence of alpha-amylase in the axis of germinating peas

α-Amylase was found in the axis portion of ungerminated pea seeds (Pisum sativum var. Alaska). The occurrence of this enzyme was demonstrated with crude homogenates (also containing β-amylase) using three different methods: the hydrolysis of β-limit dextrin, the change in absorption spectra for the iodine-starch complex, and the increase in reducing materials relative to the decrease in starch. The first method was used to quantitate the changes in α-amylase activity during germination. The increase in total amylase activity (primarily β-amylase) paralleled germination; the accumulation of α-amylase activity was not initiated for an additional day. The increased α-amylase activity was related to epicotyl growth. Approximately half of this activity was found in the etiolated stem, the distribution being higher in growing than in nongrowing portions.     

Occurrence of Conjugated Dienoic Fatty Acids in the Cellular Lipids of Pediococcus homari

Ninety-two thiolcarbamates with various substituents at the nitrogen and sulfur atoms, and their related compounds were synthesized, and their fungicidal activity against rice blast, Piricularia oryzae, and herbicidal activity against barnyardgrass, Echinochloa crus-galli, were determined in laboratory tests. The thiolcarbamate structure was necessary for the high fungicidal and herbicidal activities. The hydrophobicity of the substituents at the nitrogen atom was shown by the adaptive least-squares (ALS) method to be favorable to the fungicidal activity. The bulkier the substituents at the nitrogen atom, the less was the fungicidal activity. However, bulkiness of the substituents at the nitrogen and sulfur atoms was unfavorable to the herbicidal activity. The existence of a hydrogen atom at the nitrogen atom was favorable to fungicidal activity, but not to herbicidal activity. Correlation analyses were made to find compounds with both fungicidal and herbicidal activities against rice pests.     

网织红百分数法测定rHuEPO体内生物学活性的研究

本文通过对网织红百分数法测定rHuEPO体内生物学活性进行研究,找出网织红细胞百分数与rHuEPO浓度线性相关的范围。从而确定了用计数网织红细胞百分数的方法测定rHuEPO体内生物学活性。提出在rHuEPO体内生物学活性测定中选择合适的稀释液至关重要。对(3,3)法测定rHuEPO体内生物学活性进行了统计,批间CV、批内CV均接近10%,单次实验FL%平均数为3126%,并提出鼠间差异是造成FL%较大的主要原因;对(3,3)法与(2,2)法测定rHuEPO体内生物学活性进行比较,发现(2,2)法较(3,3)法更适合用于rHuEPO生产过程中rHuEPO体内生物学活性的测定。     

The reliability of determining the onset of medial gastrocnemius muscle activity during a stretch-shorten-cycle action

The instant at which a muscle increases its level of activity from baseline represents the onset of muscle activity. Accurate identification of muscle onset allows determination of temporal and amplitude characteristics of the surface electromyography (sEMG) signal. This investigation determined the intra- and inter-tester reliability for determining the onset of medial gastrocnemius (MG) activity using visual and automated methods. One hundred hop cycles, performed at 2.2 Hz, were selected from sEMG recordings (bandpass filtered 50–500 Hz and full wave rectified) of ten participants who performed three trials of single-leg hopping. The onset of MG muscle activity was identified by 3 separate investigators on two separate occasions and an automated method (10% of the peak activation amplitude). The duration of the anticipatory period, from muscle onset to initial ground contact, was then determined. Intra-tester (ICC from 0.72 to 0.95) and inter-tester reliability (ICC from 0.70 to 0.88) were high as was comparison to the automated method (ICC = 0.90). These findings indicate that visual onset detection was highly reproducible between testing sessions, independent investigators and comparable to an automated method. These methods may be used reliably to determine the onset of MG muscle activity during a stretch-shorten-cycle muscle action.     

Preparation of 125I-labelled monoclonal antibodies of the insulin receptor

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.