A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.     

A rapid spectrophotometric method of determining heat-resistant lysosomes in animal tissues

Rapid spectrophotometric method for the determination of thermoresistance in tissue animal lysosomes is described. The of analysis is decreased by 5-6 h, in comparison with enzymatic technique. The determination regimen was chosen in such a way that the process of lysosomal lysis was linear. The dependence of the incubation mixture temperature on the degree of lysosomal lysis was complex. The rate of lysosomal lysis rapidly increased at greater than 37 degrees C. Lysosome incubation at 0-4 degrees C for 24 h decreased its hypothermal (t = 10-30 degrees C), but not hyperthermal (t greater than 37 degrees C) sensitivity. Isolated lysosome thermoresistance may be used as an index of its stability and labialization in vivo and in vitro by various physico-chemical factors. The percentage of initial absorption (A520) and the initial rate of lysosomal lysis (delta A520/min), as well as melting temperature (Tmel) and biological half-life (t1/2) may be the measurements of such effect.     

Fluorometric method of determining the activity of ornithine decarboxylase from animal tissues

Fluorometric determination of ornithine decarboxylase activity is described. Dansyl putrescine fluorescence on TLC-plates was used to evaluate putrescine content in the samples. The dependence of dansyl putrescine fluorescence intensity on sample putrescine content was linear in the range of 0-120 nmol. Instrumental sensitivity coefficient (SF/C = 3), relative measurement sensitivity (Cmin = 0.3 nmol per sample), and basic metrological characteristics showing high reliability, accuracy and precision of enzyme activity determination were calculated. Standard error of the mean and relative standard deviation did not exceed 2.5 and 7.5%, respectively. Increased ODC activity was found in malignant and regenerating rat liver tissue, as well as in hepatomas H-27 and 48.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

Spectrophotometric method for determining the stability of lysosomes from animal tissues depending on the hydrogen ion concentration

Rapid spectrophotometric method of lysosome stability determination depending on hydrogen ion concentration is described. The time of analysis is decreased by 5-6 h in comparison with enzymic method. The process of lysosome degradation was linear at pH 6. The incubation mixture acidity dependence curve of lysosome lysis extend was complex. The lysosome lysis rate rapidly increased at pH much less than 6 less than pH. Lysosome incubation at 0-4 degrees C during 24 h decreased its sensitivity to incubation mixture acidity within the whole investigated pH range. Isolated lysosome acid resistance may be used as an index of its stability and lability in vivo and in vitro by various physicochemical factors. Percentage of initial absorbtion (A520) and initial lysosome lysis rate (delta A520/min) may be index of such effect.     

A new colorimetric method for determining the isomerization activity of sucrose isomerase

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.     

Mapping of genes determining animal quantitative traits: a method for partitioning variances

The method of quantitative trait loci (QTL) mapping based on the partitioning of variance was further developed. This extended possibilities of this method to include its use in the analysis of the data material obtained in the interbreed or interpopulation animal crosses, which provide information for genetic studies of QTL subjected to natural or artificial selection.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

Possible use of ultraviolet fluorescence for detecting protein conformational transitions in intact tissues

Spectra of fluorescence (SF) excited by the light with the wave length of 260 nm were measured with the aim of revealing the character of structural reconstructions in water-dificient plant cells. Hypocotyls of Cucumis sativus L. seedlings during soil draught served as the objects of study. With an increase of water deficiency the SF maximum is shifted to 5-8 nm to the long wave region. In wilted plant tissues free triptophane was accumulated whose SF maximum was shifted to the long wave region as compared to that of triptophane bound in proteins. The data obtained point to a limited usage of UV-fluorescence in detecting structural transitions of proteins in intact cells and tissues. In cases when the content of free triptophane is changed it is difficult to make certain conclusions about the character of conformational changes of protein macromolecules.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

Possible use of a single test culture in determining the biological activity of polyene antibiotics

A possibility using a common test-culture in estimation of biological activity of levorin, amphotericin B, mycoheptin and nistatin was studied. It was found that C. guillier mondii, strain 40, may be used as a common test-culture. It provided satisfactory microbial growth, clear and rather large inhibition growth zones with respect to all the drugs tested.