Phosphates of the naphthol AS series in the quantitative determination of alkaline and acid phosphatase activities “in situ” studied in polyacrylamide membrane model systems and by cytospectrophotometry

Summary Histochemical media for the demonstration of alkaline and acid phosphatases using phosphates of naphthol AS series as the substrates and various diazonium salts as the couplers were tested in the capability of reflecting various levels of enzyme activities.Polyacrylamide membranes with incorporated enzymes (various concentrations of purified enzymes as well as of sonicated leucocytes, macrophages and of sonicated homogenates of various organs) were used as model systems in which the activity was estimated both with biochemical and with histochemical methods. Parallel experiments were performed in sedimentation chamber preparations of guinea-pig leucocytes and macrophages in which the activity was demonstrated with the same media as in polyacrylamide films. The quantitative measurements were performed in a cytospectrophotometer using the two-wavelength method.Increasing the substrate concentration which in standard histochemical media has been 1/8 mg per ml more azo-dye is produced in the reactions for both phosphatases. If the substrate concentration is higher than 1/2 mg per ml the standard concentration of the diazonium salt (1 mg per ml) becomes insufficient for an effective capturing of the released naphthol AS in the reaction for alkaline phosphatase. Due to a very high inhibitoty effect in the case of most commercially available diazonium salts the increase of their concentration annules the beneficial action of an increased substrate concentration on the azo-dye production. 4-amino-diphenylamine diazonium sulfate has an exceptional position because it was not inhibitory even in the concentration of 4 mg/ml.In the case of acid phosphatase the higher substrate concentration was incompatible with the use of Past Red Violet LB. Hexazo-p-rosanilin was an efficient and the most chromogenic coupler used in simultaneous as well as in postincubation coupling. With the latter localization is possible on the cellular (not subcellular) level.More chromogenic combinations are generally better for the cytospectrophotometrical measurement. The shape of extinction curves of azo-dyes produced with combinations studied was similar in models and in smears. In many combinations it was dependent on the presence of lipoproteins. A too steep decline of some curves prevented the use of some combinations in alkaline phosphatase determination with the two-wavelength method, even if they are very good in the qualitative studies and might be suitable for scanning cytospectrophotometry. p]The shape of extinction curves of azo-dyes produced in the reaction for acid phosphatase using hexazo-p-rosanilin as the coupling agent was independent of the presence of lipoproteins.The curves of azo-dyes produced in simultaneous coupling are not exactly the same as the curves obtained by postincubation coupling.In receipt of a fellowship of Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Abbreviations used: AN-naphthol AS-AN phosphate; AS-naphthol AS-phosphate; B-Fast Blue B salt; BB-Fast Blue BB salt; BI-naphthol AS-BI phosphate; CL-naphthol AS-CL phosphate; DS-diazonium salt; GR-naphthol AS-GR phosphate; HP-hevazo-p-rosanilin; LB-Fast Red Violet LB salt; MX-naphthol AS-MX phosphate; S-substrate; TR-naphthol AS-TR phosphate (in the first half of the abbreviation), Fast Red TR salt (in the second half of the abbreviation); VB-4-amino-diphenylamine diazonium sulfate.     

Inhibition of Mengo virus by interferon

Gauntt, Charles J. (The University of Texas, Austin), and Royce Z. Lockart, Jr. Inhibition of Mengo virus by interferon. J. Bacteriol. 91:176-182. 1966.-The inhibition of Mengo virus replication in L cells resulting from interferon was studied quantitatively. Interferon was titrated on L cells with Western equine encephalomyelitis (WEE) virus as the challenge virus. One protective unit (PU) of interferon is the least amount of interferon which prevents cytopathic effects when a large multiplicity of WEE virus is added subsequent to overnight incubation with interferon. Ten PU of interferon reduced the yields of Mengo virus by about 90%. Larger doses of interferon, up to 220 PU, caused no further reduction in the amount of virus produced. Plaque formation by Mengo virus was also reduced in number by about 85 to 90%, but could not be further reduced. The plaques which formed on interferon-treated cells were reduced in size. We were unable to obtain a virus population with increased resistance to interferon action by use of five successive growth cycles in interferon-treated cultures. Analysis of the cell population for the proportion of cells able to act as infectious centers revealed that incubation of cells with 10 PU of interferon decreased the proportion of virus-yielding cells by 80%. The yield of virus per virus-producing cell was decreased by 40 to 60%. Despite the reduction in yields, plaques, and infectious centers resulting from interferon, all doses of interferon failed to prevent the complete destruction of the cells. Experiments with puromycin indicated that the cytopathic effects observed in L cells infected with Mengo virus required that a virus-directed protein be synthesized between 4 and 5 hr postinfection. The evidence suggested, therefore, that the Mengo virus genome was able to code for new protein synthesis in the absence of the production of infectious virus.     

Repair of thermal injury of Staphylococcus aureus

Iandolo, John J. (University of Illinois, Urbana), and Z. John Ordal. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142. 1966.-Exposure of Staphylococcus aureus MF 31 to sublethal temperatures produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 55 C for 15 min, more than 99% of the viable population was unable to reproduce on media containing 7.5% NaCl. The data presented demonstrate that thermal injury, in part, occurred owing to changes in the cell membrane, which allowed soluble cellular components to leak into the heating menstruum. When the cells were placed in a limiting medium, complete recovery did not occur, regardless of the incubation time. The temperature and the pH which produced the optimal rate of recovery were similar to those described previously for the multiplication of uninjured cells. However, the rate of recovery as well as the unchanging total count during recovery indicated that cell multiplication was not a factor during the recovery process. The nutrient requirements for the complete recovery of injured cells consisted of a solution containing an energy source, such as glucose, a mixture of amino acids, and phosphate. The use of the metabolic inhibitors, penicillin, cycloserine, 2,4-dinitrophenol, and chloramphenicol, did not inhibit recovery. Actinomycin D, however, completely suppressed recovery. This result implied that ribonucleic acid synthesis was particularly involved; this inference was substantiated by radio tracer experiments. The rate at which label was incorporated in the nucleic acid fraction paralleled that of recovery and the return of salt tolerance.     

Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.