Effect of N-tris (Hydroxymethyl) Methylglycine and N-2-Hydroxyethyl-piperazine-N'-2-Ethanesulfonic Acid Buffers on Linolenic Acid as a Substrate for Flaxseed Lipoxidase

The delay in, or loss of, flaxseed lipoxidase activity in N-tris (hydroxymethyl) methylglycine and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffers with linolenic acid as a substrate appears due to an alteration of the lipid micelle. Flaxseed lipoxidase activity is dependent on the ionic strength of the assay solution. These effects are not observed with linoleic acid as substrate. The influence of these 2 buffers on linolenic acid micelles may have a direct bearing on recent reports of chloroplast structure and activity in these buffers.     

Singlet oxygen generation by the lipoxidase system

The rate of cytochrome c bleaching by the lipoxidase system is increased in deuterium oxide buffers. This result is consistent with the view that the lipoxidase system produces singlet oxygen. This species does not seem to be responsible for the self-catalyzed destruction of lipoxidase.     

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase,platelet aggregation and lipoxidase

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants α-naphthol, guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-α-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited specifically by α-naphthol, guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-α-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus α-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-α-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.     

Differential inhibitory effects of vitamin E and other antioxidants on prostaglandin synthetase, platelet aggregation and lipoxidase.

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from the bovine vesicular gland is inhibited by the antioxidants alpha-naphthol. guaiacol, NDGA and propyl gallate. Prostaglandin biosynthesis in this system is not inhibited by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. Arachidonic acid induced platelet aggregation is inhibited by specifically by alpha-naphthol. guaiacol, NDGA and propyl gallate. Both arachidonic acid induced platelet aggregation and ADP induced platelet aggregation are inhibited non-specifically by the antioxidants BHT, DL-alpha-tocopherol and Trolox C. All antioxidants tested in this study inhibit soybean lipoxidase. Thus alpha-naphthol, NDGA and propyl gallate are non-specific inhibitors of both prostaglandin synthetase and soybean lipoxidase while BHT, DL-alpha-tocopherol and Trolox C are specific inhibitors of soybean lipoxidase alone.     

无腥味大豆种质创新与利用

大豆是植物油和植物蛋白的主要来源,但大豆蛋白中含有3种结构不同的脂肪氧化酶同功酶,是产生豆腥味的根源,使大豆利用及加工受到限制。从1994年开始我们进行大豆脂氧酶缺失基因的转育与种质创新研究,目前获得了一批综合农艺性状优良的低腥味材料,其中绥无腥豆1号已于2002年3月通过黑龙江省农作物品种审定委员会审定,并开始应用于生产和加工领域。     

On the presence of proteolytic activity in glycosaminoglycan-degrading enzyme preparations

A solid-phase protease assay has been used to screen different commercial preparations of glycosaminoglycan-degrading enzymes for the presence of proteolytic activity. Proteases cannot be detected in preparations of testicular hyaluronidase and of chondroitinase at the concentration used for histochemical purposes. Commercial Streptomyces hyaluronidase contains proteolytic contaminants detectable at the concentration used for histochemistry. At higher concentrations, all preparations appear to be contaminated with proteases. The results obtained using this assay suggest that addition of a mixture of proteinase inhibitors containing N-ethylmaleimide, EDTA, pepstatin, and phenylmethanesulfonylfluoride or soybean trypsin inhibitor has little effect on the proteolytic activity of the glycosaminoglycan-degrading enzyme preparations, irrespective of the pH used. Moreover, the use of EDTA in this mixture is questionable. This study also describes two testicular hyaluronidase preparations that may be particularly useful in functional studies of the living organism, as they are only slightly contaminated.     

Light-dependent modifications in the metabolic responses of squash seedlings

Light-induced modifications in lipoxidase metabolism and chlorophyll formation in the cotyledon of squash (Cucurbita moscata) seedlings were determined. The enzyme activity decreased as light intensity increased, but chlorophyll continued to accumulate long after lipoxidase activity had virtually disappeared. Considering the differences in the levels of irradiance required to manifest the optimal responses, and also from the results obtained with ultraviolet and red, far-red light treatments, any causal relationship between lipoxidase disappearance and chlorophyll synthesis was ruled out.

The observed light-saturation phase in the chlorophyll synthesis, indicated that chlorophyll formation was initially controlled by the phytochrome system. No similar saturation stage for the enzyme responses was observed.

The sensitivity of lipoxidase to prolonged light exposures suggested a strong correlation with the known photoreactions presumed to be controlled by the high energy reactive-phytochrome system. Lipoxidase metabolism is, therefore, suggested as a biochemical index for the photomorphogenic reactions similar to the ones induced by the high energy reaction.

     

Catalase-Aminotriazole Assay, an Invalid Method for Measurement of Hydrogen Peroxide Production by Wood Decay Fungi

The catalase-aminotriazole assay for determination of hydrogen peroxide apparently cannot be used for measuring hydrogen peroxide production in crude preparations from wood decay fungi because of materials in the crude preparations that interfere with the test.     

Inactivation of slow reacting substance of anaphylaxis (SRS-A) by lipoxidase.

Lipoxidase (E.C.1.13.1.13) inactivates rat SRS-A. The inactivation is time, temperature, and concentration-dependent. Linoleic acid and eicosatetraynoic acid inhibit the enzymatic deactivation of the mediator. It is concluded that SRS-A is a genuine substrate for lipoxidase and contains a cis, cis-1, 4-pentadiene structure. It is suggested that lipoxidase could play a major role in tissue removal of the mediator during hypersensitivity reactions.     

Inactivation of slow reacting substance of anaphylaxis (SRS-A) by lipoxidase

Lipoxidase (E.C.1.13.1.13) inactivates rat SRS-A. The inactivation is time, temperature, and concentration-dependant. Linoleic acid and eicosatetraynoic acid inhibit the enzymatic deactivation of the mediator. It is concluded that SRS-A is a genuine substrate for lipoxidase and contains a cis, cis-1, 4-pentadiene structure. It is suggested that lipoxidase could play a major role in tissue removal of the mediator during hypersensitivity reactions.     

Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays

A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations exhibiting discordant relative activities in the different assay systems. Its relative in-vivo activity was almost 50% lower than that found in the 3 other assays. This could be due to denaturation of the hormone during its preparation or to selection of isoform(s) not representative of the whole population of molecules. For standardization of horse CG preparations by in-vivo assay, IRP2 has proved to be a reliable standard. However, for the standardization of preparations by in-vitro methods, a standard giving consistent results in in-vivo and in-vitro assays must be used. The present report indicates that the NIH standard, but not IRP2, fulfils these requirements.     

A high-throughput hybridization method for titer determination of viruses and gene therapy vectors.

One of the challenges facing researchers working with viruses and gene therapy vectors is the need to rapidly assay for infectious virus. Current methods used to titer many viruses are cumbersome and are not amenable to handling large numbers of samples. Here we describe the development of an assay that can rapidly quantify infectious viruses and gene therapy vectors. The assay relies on biological amplification of viral sequences and hybridization of labeled probes to immobilized nucleic acid from infected cells. The amplification of the viral genome makes this a highly sensitive method. The assay is configured in a high-throughput format that has been used to detect recombinant adeno-associated virus (AAV), wild-type AAV and infectious adenovirus. The assay is quantitative, and can be used to titer virus preparations with or without a known standard.     

Innocuity testing of foot-and-mouth disease vaccines. II. Aziridine-inactivated antigen produced in baby hamster kidney cells

Methods for the testing of preparations of aziridine-inactivated foot-and-mouth disease virus for the absence of infective particles were studied. The system used for virus production, suspension cultures of baby hamster kidney cells, proved to be the most sensitive detection system for traces of infective virus as long as the 146S antigen concentration was below 1 microgram per 10(6) cells. Above this level interference may mask the presence of non-inactivated virus. Thus in a 1-1 suspension culture 1 mg of inactivated 146S antigen equivalent to at least 300 doses of vaccine could be tested. The kinetics of inactivation may be studied by the agar-cell suspension plaque assay which is nearly equal in sensitivity to the method described above. Antigen concentrations at which interference occurred were also estimated for this type of assay. Inactivation of polyethylene glycol-concentrated virus showed 'tailing-off' and such virus preparations should not be used in vaccine production. The data are discussed with reference to the recommendations for innocuity testing in the European Pharmacopoeia.     

Studies on the characterization of bovine adipocyte precursor cells and their differentiation in vitro, using an indirect-labelled-second-antibody cellular immunoassay

Antisera with specific reactivity towards adipocyte cell surfaces were prepared and characterized. These preparations, absorbed to remove reactivities toward other cell types were used in an indirect labelled second antibody cellular immunoassay which distinguished bovine adipocyte precursors from fibroblasts and which allowed the progress of precursor cell differentiation in culture to be monitored with precision and sensitivity. The assay could be modified to use fluorescent, rather than radioactively-labelled, second antibody preparations and the changing reactivity of differentiating precursors to be visualized. Labelling of preparations in this way also allowed precursor cell populations to be analysed and quantitated using fluorescence-activated cell sorting technology.     

Neurovirulence tests of three 17D yellow fever vaccine strains

The results of tests of the neurovirulence of three yellow fever vaccine preparations of different lineages are presented. Two preparations that have been used to make vaccines of acceptable safety and efficacy gave very similar results. A third preparation from the Robert Koch Institute, designated 168-73, was proposed as a reference preparation for the mouse potency assay in 1985 by WHO, but has been more often used as a reference in the monkey neurovirulence test. In the test described here 168-73 was of lower virulence than either of the other two preparations.     

Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations

Cell viability in probiotic preparations is traditionally assessed by the plate count technique. Additionally, fluorescent staining combined with epifluorescence microscopy or flow cytometry has been developed for the viability assessment, but the currently available assays are either laborious or require highly sophisticated equipment. The aim of this study was to investigate the applicability of a microplate scale fluorochrome assay for predicting the cell state of freeze-dried Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis preparations. In addition to viability assessment with LIVE/DEAD BacLight Bacterial Viability Kit, DiBAC(4)3 stain was used for the kinetic measurement of changes in bifidobacterial cell membrane functions during exposure to low pH. The microplate scale fluorochrome assay results on the viability and cell numbers of probiotic preparations correlated well with the results obtained with the culture-based technique and (with few exceptions) with epifluorescence microscopy. The assay was applicable also for the viability assessment of stressed (acid-treated) cells provided that the cell density in treatments was adjusted to the optimal measurement level of the fluorometer. The microplate scale fluorochrome assay offers a rapid and robust tool for the viability assessment of probiotic preparations, and enables also kinetic measurements.     

A Colorimetric Assay for Cytokinin Oxidase

A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal andp-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.     

Determination of high-affinity oestrogen-receptor sites in uterine supernatant preparations

An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k(-1)) and low-affinity (k(-2)) receptor sites and [(3)H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.     

Measurement of beta-galactosyltransferase activity in cell extracts with an ELISA-based assay

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal:N-acetylglucosamine beta-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC11 cells (a mutant of the CHO cell line, Pro -5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Gal beta 1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The beta-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 beta-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare beta-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro-5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.