New and simple plate test for screening relative transfructosylation activity of fungi

Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase and/or fructofuranosidase activities. However, the search for other fungi with higher transfructosylation activity remains a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed which involved the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the (a) glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and (b) fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the fungal colony. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity and also gives a relative quantitative idea of the transfructosylation activity of different fungi species.     

Micromethods for the study of hexokinase in human erythrocytes

A direct radioassay for the erythrocyte enzyme using U14C-glucose as substrate has been developed. With respect to the indirect spectrophotometric assay this method allows for the determination of true hexokinase activity. The assay proposed is sensitive, rapid and well suited for the determination of hexokinase activity in the erythrocyte lysate where the enzyme level is particularly low.     

A new colorimetric determination of D-amino acid oxidase and urate oxidase activity.

A new method of colorimetric determination of d-amino acid oxidase and urate oxidase using catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole is reported. This method is based on the combination of two steps of enzyme reactions and colorimetric procedure. The values obtained by this method are satisfactorily correlated with those obtained by the dinitrophenylhydrazine method for d-amino acid oxidase activity and the ultraviolet method for urate oxidase activity and showed good reproducibility and accuracy. It is considered that the method can be useful as a method of activity determination for studying enzyme kinetics and the reaction mechanism.     

D-核糖产生菌转酮醇酶活性分析

芽孢杆菌属的转酮醇酶(Transketolase,TKT)变异株可利用培养基中的D-葡萄糖,将其转化成D-核糖分泌于细菌培养基中。阐述了D-核糖产生菌转酮醇酶的活性测定方法,制备了测定所必需的2种酶。     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.     

Determination of rifamycin B activity in culture liquids and in preparations with varying degrees of purity]

A possibility of using the biological method of rifamycin B activity determination in the fermentation broth and dry preparations of various purity levels was studied. It was found that the biological method was useful only for determination of rifamycin B activity in preparations containing not less than 850 gamma/mg of the main product. When the activity of rifamycin B was determined in the fermentation broth and crude preparations containing less than 800 gamma/mg of the main product, the results of the biological assay were always higher as compared to those of spectrophotometrical estimation. It was accounted for the effect of other rifamycin types possessing high biological activity.     

Determination of angiotensin-converting enzyme activity using dansyl-Phe-Ala-Arg as the substrate

Modificated method for the determination of the angiotensin converting enzyme (EC 3.4.15.1) activity in neural tissue is proposed. The methods is based on fluorimetrically determination of released dansyl-Phe from dansyl-Phe-Ala-Arg at pH 7.6. Km is 50 +/- 10 MM. The high specificity of the method is provide by using captopril, the high specific angiotensin converting enzyme inhibitor. The sensitivity of the method is 0.001 nmol/min per mg protein that it is lesser in 15-fold than the lowest enzyme activity in brain region. The distribution of the angiotensin converting enzyme activity that using the method in brain regions and peripherial tissues of rat is presented.     

Combined Ki-67 and feulgen stain for morphometric determination of the Ki-67 labelling index

In this note we present a combined Ki-67 and Feulgen stain for morphometric determination of the Ki-67 labelling index. The immunohistochemical part of this double staining technique is based on the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method, visualizing the enzyme activity by the nitro-blue-tetrazolium chloride (NBT)/bromo-chloro-3-indolyl-phosphate (BCIP) technique. The NBT/BCIP complex resists the hydrolytic activity of the Feulgen stain. The staining method presented allows semi-automatic determination of both the total nucleus-area as well as the Ki-67 positive nucleus-area using a morphometric computer system. The Ki-67 labelling index thus achieved is based on the relative nuclear area of Ki-67 positive nuclei and is clearly more precise and efficient than the counting method using an ocular grid.     

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.     

Use of the passive hemagglutination inhibition reaction for the purpose of determining the antigenic activity of anatoxins in vitro]

The work deals with the study of the possibility of using the passive hemagglutination inhibition test (antibody neutralization) for the determination of the antigenic activity of botulinum toxoids, types A, B and E. Erythrocytic diagnostic preparations were shown to allow the determination of up to 0.1 g of antigen. At the same time in vitro determinations were found to satisfactorily correlate with the results of in vivo determinations of the antigenic activity of these toxoids in the antitoxin-fixation test.     

Polarographic determination of esterase and lipase activities using esters of -naphthols

The polarographic method of the determination of esterase and lipase activities using esters of β-naphthol has been elaborated. The activity of esterase of pig liver, house-fly heads, lyophilized horse serum, and human serum; and lipase activity of pig liver and pancreas was determined. Advantages of the polarographic method lie in using the same calibration curve of nitrosated β-naphthol for determinations of activities of several enzyme sources and in the stability of nitrosated β-naphthol in comparison to the colored reaction product of β-naphthol and tetraazotized o-dianisidin needed for spectrophotometry.     

极谱氧电极法测定超氧化物歧化酶活性的改进

对SOD的极谱氧电极测定法做了如下修改:a.室温测定,b.酶活性用标准SOD标定,c.反应在磷酸缓冲液中进行,d.增大邻苯三酚的用量.改进后克服了易在电极薄膜表面产生气泡等问题,测定灵敏度及线性范围增大.     

Antioxidant and antiulcer activity of aqueous extract of a polyherbal formulation

The aqueous extract of Hingwashtak churna was evaluated for gastroprotection in rats using the ibuprofen and ethanol induced ulcer models. Efficacy was assessed by determination of mean ulcer size, ulcer number and ulcer index. Oral administration of the aqueous extract (750 mg/kg) significantly protected against gastric lesions by 84.96% and 91.12% as compared to ranititidine (95.54 and 95.2%) in the ibuprofen and alcohol induced ulcer models respectively. The findings suggest that the significant gastroprotective activity could be mediated by its antioxidant activity which was evaluated by using different antioxidant models of screening.     

Determination of urease activity by thermal conductivity gas chromatography

A rapid, simple and inexpensive procedure for the determination of urease activity by using a thermal conductivity gas chromatography method is presented. The procedure is based on the determination of CO2 released in the urease reaction, and showed low coefficient of variation (c.v. less than 1%) and high sensitivity (detection limit 10(-12) mol). This procedure is also suitable for determination of other decarboxylating enzyme activities.     

Beta-mannosidase of trophoblast and humana skin fibroblasts]

Conditions for assay of beta-mannosidase activity in human chorionic villi were studied using the fluorogenic substrate 4-methylumbelliferyl-beta-D-mannopyranoside. Comparison of the biochemical properties of the chorionic villi beta-mannosidase with those of the enzyme from human cultured fibroblasts showed their similarity. Like the enzyme from skin fibroblasts, the chorionic villi beta-mannosidase had rather high activity. Both enzymes had virtually the same pH optimum (4.2-4.7) and Km value. The data presented suggest that chorion biopsy specimens can be used for prenatal determination of beta-mannosidase activity at the early stage of development.     

Induction of Ears from Maize Seeds in Vitro and Plant Regeneration from Ovaries of Unfertilized Ears

A new colorimetric method for determining the isomerization activity of sucrose isomerase was developed. This colorimetric method is based on the enzymatic reactions of invertase and glucose oxidase-peroxidase (GOD-POD). The main scheme for assaying sucrose isomerase activity is to degrade sucrose in the reaction mixture to glucose and fructose by invertase and to detect the concentration of glucose generated using GOD-POD. The concentrations of trehalulose and isomaltulose, reaction products of sucrose isomerase, are calculated from the concentration of glucose. This method allows rapid and accurate determination of the isomerization activity of sucrose isomerase without inhibition by hydrolysis activity.     

Simple and non-radioactive method for determination of sialyltransferase activity

A new, non-radioactive and cheap colorimetric method for determination of activity of sialyltransferases of various specifities using natural substrates based on 2-thiobarbituric acid assay is presented. The assay was tested with three different sialyltransferases (a-2,3 and a-2,6) and compared with the radioactive assay.     

The activities of carbonic anhydrase and alkaline phosphatase in ancient human bones. Purification and characterization of outer peripheral, cytosolic, inner peripheral, and integral CA

In the present study, bone carbonic anhydrase was isolated from ancient human bones and its characteristic features were determined. For this purpose, the skull bone of about 3000 years age was used. The purification was performed in four steps. Four different isoenzymes of CA, including outer peripheral, inner peripheral, integral, and cytosolic were purified and characterized. Affinity chromatography using Sepharose-4B-L-tyrosyn sulfanilamide as a support material was used in its purification. Two different methods were used for enzymatic activity determination: a) hydratase, and b) esterase methods. Bradford and Coomassie Brillant Blue methods were used for protein determination. Optimal pH, temperature, and molecular weight determinations were performed by conventional methods. The purification degree and the subunits, if present, were determined by SDS-PAGE. The effects of some chemicals on the enzyme were also investigated. The most cardinal finding was that the enzymatic activity has been found in antique human bone, showing some other enzymatic activity. That the alkaline phosphatase activity has been determined in the same sample supports the finding of carbonic anhydrase.     

Direct quantification of dimethylsulfoniopropionate (DMSP) in marine micro- and macroalgae using HPLC or UPLC/MS

A simple method for the direct quantification of dimethylsulfinopropionate (DMSP) using HPLC or UPLC coupled to UV and/or MS detection is introduced. The protocol is applied for the determination of DMSP from marine micro- and macroalgae. The method is based on the derivatisation of DMSP using 1-pyrenyldiazomethane followed by reversed phase HPLC or UPLC separation. The detection limit is 590 nM, corresponding to 1 ng DMSP per injection. Using a combination of UV and MS detection the calibration curves were linear in the range of 2.93 microM to 11.7 mM concentrations. We show that direct determination of DMSP is possible from macroalgal tissue and microalgal cultures if DMSP-lyase activity is suppressed during work-up.     

Anticonvulsant and analgesic activities of crude extract and its fractions of the defensive secretion from the Mediterranean sponge, Spongia officinalis

ABSTRACT: This study progresses in the direction of identifying component(s) from the Mediterranean sponge, Spongia officinalis with anticonvulsant and analgesic activities. We investigated the efficacy of crude extract and its semi-purified fractions (F1-F3) of the defensive secretion from Spongia officinalis for their in vivo anticonvulsant activity using the pentylenetetrazole (PTZ) seizure model and analgesic activity using the writhing test in mice. Among the series the crude extract exhibited interesting analgesic activity in a dose dependent manner. Similarly the fraction F2 showed a partial protection of mice from PTZ-induced seizure and interesting analgesic activity in a dose dependent manner. The purification and the determination of chemical structure(s) of compound(s) of this active fraction are under investigation.