Absorption-Spectrophotometric Determination of DNA (Deoxyribonucleic Acid) in Milk Ad Modum Feulgen

The Feulgen reaction is examined by absorption photometric measurements at 565 nm with correction for varying background absorption at 485 nm. This correction is done to improve the accuracy of measuring. Examinations of the accuracy of analysis for the Feulgen reaction and the direct microscopic cell counts show that the former is of the same order, when the cell count is made at a working factor of 17,000. The standard deviations expressed in percentage of the reaction value are greater the lower the reaction is, whereas the absolute standard deviation is reduced. It has been demonstrated that addition of a few (< 5) µg of DNA per ml milk can give Feulgen reaction. The coefficient of correlation between log added amount of DNA and log Feulgen reaction is 0.98. The coefficient of correlation between log Feulgen reaction and log cell content depends on the accuracy at which both determinations are made. In a determination of cell content at a working factor of 550, and several determinations of the Feulgen reaction the coefficient of correlation (r) is found to be 0.98. A cell content determination at a working factor of 20,000 and a single determination of Feulgen reaction on each milk sample yields r = 0.83. An examination of foremilk samples of the same cell content reveals on an average the highest reaction in mastitis-affected quarters which have been infected within the last 4 weeks. Quarters that have been infected for more than 4 weeks, on an average show the second highest reaction, whereas quarters of physiological cell number show the lowest reaction. The DNA-content in most cases is found to be too high compared with the number of cells counted microscopically. The DNA-content in centrifugated cells corresponds to the one calculated theoretically. The surplus DNA-content can be demonstrated in the cell-free skim milk fraction, probably originating from destroyed cells. The studies performed suggest that determination of the content of 2-deoxyribose in milk by means of the Feulgen reaction is a more correct measure of the cell content in the milk than is the microscopic cell count. Studies are being continued for illustration of these conditions.     

The photometric determination of gangliosides with the sulfo-phospho-vanillin reaction

A simple, quantitative method is described for the photometric determination of gangliosides. The procedure is based on the sulfo-phospho-vanillin reaction, and does not require prior hydrolysis. It has been shown that the reaction is probably due to oxidation by sulfuric acid of the sphingosine moiety which results in the formation of aldehydes or ketones or both which then react with the phosphoric acid-vanillin reagent to produce a rose-colored complex. The reaction permits the determination of the amount of ganglioside present in a sample; and, together with the resorcinol reaction to measure the NANA content, it can be used to determine whether a purified ganglioside is a mono-, di-, or trisialoganglioside.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

The use of the o-phthalaldehyde reaction as a sensitive assay for protein and to determine protein in bacterial cells and dental plaque

Alkaline hydrolysis of protein followed by reaction with o-phthalaldehyde has permitted the determination of less than 10 ng of protein fluorometrically. When intact proteins were analyzed with o-phthalaldehyde the reaction was less sensitive. This reaction has been applied to analysis of the protein content of dental plaque and bacterial cells.     

Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.     

4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.     

Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - from the field to the test tube and back

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.     

Evidence of the heterogeneity of mast cell population based on their biogenic amine content

The heterogeneity of the mast cells localized in various organs has been demonstrated by histochemical determination in their indole amine and histamine content. The strongest amine reaction was found in the mast cells in the mesenterium and peritoneal fluid where their maturation occurs. The reaction of the subcutaneous connective tissue is weaker; in the thyroid gland the reaction is weak as well as diffuse. In the thymus the mast cells localized along the vessels give a strong histamine reaction. The experiments support previously published data concerning the heterogeneity of mast cell populations.     

Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.     

Adaptation and constraint in the evolution of environmental sex determination

When environments differentially influence male and female performance, environmental sex determination (ESD) might evolve. The conclusion from several previous theoretical models was that reaction norms for sex determination should have a single, sharp threshold, with only females being produced in some environments and only males in others. These reaction norms can be disadvantageous in fluctuating environments, however, because they lead to sex-ratio fluctuations. We analysed the evolution of ESD, looking for equilibrium strategies in unconstrained as well as constrained strategy spaces. We identified situations where a single-threshold reaction norm is not evolutionarily stable. In these cases, we found stable strategies in the form of complex reaction norms, showing an oscillatory pattern of sex determination with respect to variation in an environmental variable. Considering that constraints could prevent such phenotypes from being realized, we found that certain randomized reaction norms, with probabilistic sex determination for a range of environments, would achieve nearly the same fitness. We also investigated reaction norms constrained to have a single threshold and found that genetic polymorphism in the environmental threshold value could evolve, producing a similar effect as a randomized reaction norm. We argue that the appearance of genetic variation can be regarded as an alternative outcome when constraints prevent the evolution of a more complex or a randomized strategy.     

Simultaneous determination of zymogen activation time and zymogen level using chromogenic substrates in blood coagulation analysis

The amidolytic activities of plasma generated by means of thromboplastin and Ca++, on the one hand, and by means of partial thromboplastin, a contact activator and Ca++, on the other hand, were determined using synthetic, chromogenic factor Xa substrates with low affinity for thrombin (CH3SO2-D-Leu-Gly-Arg-pNA and CH3SO2-D-Nleu-Gly-Arg-pNA). In this way, the activation process by splitting off the p-nitroaniline was followed. Besides the summary detection of factor Xa was obtained after addition of hirudin. During preincubation with partial thromboplastin and contact acti (Actin) in Ca++-free medium, an amidolytic activity so far unidentified was generated that renders evaluation of the activation process difficult. In the test system with partial thromboplastin, factor Xa could not be determined and the thrombin-like activity that can be inhibited by hirudin did not correspond to the amount of prothrombin present in plasma. In contrast, activation of factor X and prothrombin by thromboplastin and Ca++ could be followed and the content of the two zymogenes could be detected simultaneously. In general, under optimized reaction conditions, automated systems might be developed that would provide additional diagnostic information about determination of clotting time, on the one hand, and about quantitative determination of zymogen, on the other hand.     

Immune reaction of tumor-bearing mice to Propionibacterium acnes and the antitumor effect of the bacteria

Summary The relationship between immunological reaction to Propionibacterium acnes (PA) and the antitumor effect of the injected bacterium was investigated. The aim was to determine whether the strength of the immune reaction to the bacterium can be used to predict its antitumor effectiveness. C3Hf/Sed mice received SC injections (right thigh) of viable cells of a methylcholanthrene-induced fibrosarcoma. When the tumor grew to 5 mm, the hosts received 350 g PA IV as the antitumor treatment. Cellular immunity (footpad test) to PA was assayed in one group of these mice 14 days later, and in the other anti-PA agglutinins were determined 28 days later. The PA injection cured 22 of 58 mice in the first, and 20 of 46 mice in the second group. Footpad reaction and agglutinin titers to PA in cured mice were not statistically different from those in mice eventually killed by the tumor. Therefore, the strength of the immune reaction to PA in tumor-bearing mice could not be used to predict the antitumor effectiveness of the bacterium.Andres Soriano, Director of Cancer Management     

The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples

We present a fast to perform spectrophotometric method for the quantification of ascorbic acid and its oxidized form dehydroascorbic acid in biological samples. The assay detects a chromophore formed during the reaction of dehydroascorbic acid with methanol in phosphate/citrate buffer. This reaction can also be employed for the determination of ascorbate (vitamin C) in the presence of ascorbate oxidase. The major advantage of the developed protocol for the determination of both forms of vitamin C is a simple spectrophotometrical single end point determination. It is demonstrated that the methanol method is an improvement compared with a commercially available test kit for the determination of vitamin C. Using the methanol method, a dose-dependent increase in intracellular ascorbic acid was determined upon incubation of L-929 cells and RAW 264.7 macrophages with increasing concentrations of extracellular ascorbate. In blood serum, vitamin C was determined at concentrations between 46 and 97 microM. Supplementation with different amounts of ascorbate showed satisfying recovery. In L-929 cells, even unphysiologically high amounts of reactive nitrogen species were unable to completely oxidize intracellular vitamin C.     

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.     

Structure and carbohydrate histochemistry of the stomach in eight species of teleosts

The histology and carbohydrate histochemistry of eight teleostean stomachs are compared. Three gross anatomical types of stomachs are described and their shapes appear to correlate somewhat with feeding habits. Each type can be divided histologically into a corpus and pylorus. Gastric glands, containing only one cell type, occur in the copora of all species, but are present in the pylori of esocids only. As a single cell can produce both enzymes and hydrochloric acid such cells may be comparable to those of amphibians but not mammals. Lamina propria and submucosa are indistinctly separated in corpora but better defined in pylori by an intervening muscularis mucosa. The arrangement of the muscularis into inner circular and outer longitudinal layers is the opposite of that seen in the esophagus. Gastric mucous cells show species variations in localization of epithelial mucosubstances, which in broad terms are recognized as sulfomucins, sialomucins and neutral mucosubstances. A piscivorous diet does not appear to demand any particular type of carbohydrate. Within the Centrarchidae, gastric pit cells vary in carbohydrate content from only neutral mucosubstance to only weakly acidic sulfomucin; two species contain both types. A positive PAS reaction on the surface of gastric epithelial cells is suggestive of a striated border and thus possibly absorptive function. The absence of stomachs in some teleosts and the evolutionary and dietary significances are discussed.     

Fluorogenic substrate [Ala-Pro]2-cresyl violet but not Ala-Pro-rhodamine 110 is cleaved specifically by DPPIV activity: a study in living Jurkat cells and CD26/DPPIV-transfected Jurkat cells.

Fluorogenic substrates [Ala-Pro](2)-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro](2)-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro](2)-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro](2)-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro](2)-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction.     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

分泌性的糖蛋白Wnt5a对永生化小鼠黑素细胞系melan-a细胞产黑素的影响

目的：探讨分泌性的糖蛋白Wnt5a对永生化小鼠黑素细胞系melan-a细胞产黑素的影响。方法：用带有Wnt5a基因的腺病毒及对照组腺病毒作为载体感染体外培养的melan．a黑素细胞;MTT法测定细胞增殖率;体外氧化DOPA反应法测定酪氨酸酶活性;NaOH法测定黑素含量;RT-PCR方法检测melan-a细胞中MITF的表达。结果：与AdGFP对照组相比,AdWnt5a处理组melan-a细胞的增殖率明显降低（P〈0．01）;DOPA反应法和NaOH法检测结果发现,Wnt5a能显著降低melan-a细胞内酪氨酸酶活性（P〈0．01）以及黑素含量（P〈0．05）;RT—PCR结果表明,wnt5a显著下调melan—a细胞内MITF的表达（P〈0．01）。结论：以上结果显示,AdWnt5a处理组melan-a细胞的增殖率、酪氨酸酶活性、产黑素的量及MITF的表达均有所下降。实验结果提示,Wnt5a能有效抑制melan-a细胞产黑素的能力,并且其作用机制可能与下调MITF的表达有关。