ALBERT

Notes: SYNOPSIS. By means of the ninhydrin-Schiff method for proteins a diffuse reaction as well as one localized in granular inclusions can be shown in the cytoplasm of fibroblasts, epithelial cells, and macrophages in trypsin-dispersed chick liver cell cultures. Nuclei and nucleoli also take the specific stain. A progressive loss of cytoplasmic and nuclear staining occurs in the fibroblasts in cultures infected with a relatively pathogenic strain of T. vaginalis. A loss occurs in epithelial cells in advanced stages of degeneration, but in less damaged cells, while the diffuse reaction disappears, the number and staining intensity of the cytoplasmic inclusions remain unchanged or possibly may increase somewhat. The intensity of the diffuse reaction and the number and size of the characteristic inclusions increase in the active, parasite-free, experimental macrophages, but phagocytes with trichomonads closely applied to their external surfaces and those containing the flagellates within their cytoplasm typically retain only a few weak-staining inclusions.Similar distribution of alkaline and acid phosphatases occurs in preparations treated according to Gomori's and Burstone's methods, except that no nuclear staining is obtained with the latter. Activity of both enzymes is localized primarily in inclusions which are dispersed thruout the cytoplasm of fibroblasts and epithelial cells and tend to accumulate along the cell membranes and around the nuclei. In the course of infection with T. vaginalis there is a progressive loss of alkaline phosphatase from both cell types; however, the acid phosphatase activity increases. In the control macrophages both enzymes are localized in mostly rather large, rounded cytoplasmic inclusions. The number of such inclusions increases in the parasite-free experimental macrophages, but only a few weak-staining granules remainin phagocytes with engulfed trichomonads and in those whose external surfaces are in direct contact with the parasites. The loss of the inclusions is less apparent in macrophages containing degenerated flagellates than in the ones with healthy trichomonads, but regardless of the condition of the parasites, the highest enzymatic activity is found around them.ATPase and 5′-nucleotidase are localized in small granules dispersed thruout the cytoplasm of fibroblasts and epithelial cells. The granules tend to accumulate along the periphery of the cells and around the nuclei. A diffuse cytoplasmic reaction is present in preparations processed for 5′-nucleotidase. Nuclei and nucleoli give positive reactions for both enzymes. In the course of infection with trichomonads, activity of the 2 enzymes declines in both culture cell types. Control macrophages have diffuse cytoplasmic reaction for ATPase and 5′-nucleotidase and these enzymes are localized also in rounded cytoplasmic inclusions. Activity of both enzymes increases in the parasite-free experimental phagocytes, but little if any diffuse staining and only a few characteristic inclusions are left in macrophages with engulfed healthy trichomonads and in those whose external surfaces are invested with the flagellates.The ninhydrin-Schiff-positive inclusions found in the macrophages appear to be the same as some of those which have acid phosphatase activity and may well be identical with the glycolipoprotein bodies noted by us previously. On the grounds of their chemical constitution and behavior it seems likely that the inclusions are lysosomes.

Notes: SYNOPSIS. In trypsin-dispersed chick liver cell cultures malic dehydrogenase activity is localized in granules distributed thruout the cytoplasm of fibroblasts, epithelial cells, and macrophages. A progressive increase of the enzymic activity in all cell culture elements, except for phagocytes whose external or internal surfaces remain in direct contact with the parasites, accompanies infection of the cultures with a relatively pathogenic strain of Trichomonas vaginalis. In such phagocytes most staining for malic dehydrogenase is lost. Epithelial cells and parasite-free macrophages in experimental cultures also have a diffuse cytoplasmic reaction. No lipase activity is present in fibroblasts, but epithelial cells and macrophages in chick liver cell cultures contain numerous reactive granules. A strong diffuse cytoplasmic reaction is found in the epithelial cells and a weaker one in the control phagocytes. In cultures infected with T. vaginalis the enzyme is lost progressively from the epithelium and from those macrophages which have engulfed the parasites or whose external surfaces are invested with the flagellates; however, no significant changes in lipase activity can be found in parasite-free experimental phagocytes. In all cell types found in chick liver cultures, the reaction for nonspecific esterase is localized in cytoplasmic inclusions of varying size, some of which tend to accumulate along the cell membranes and around the nuclei. In addition, a weak diffuse cytoplasmic reaction is seen in the epithelial cells. Most cells in cultures infected with T. vaginalis have a significant increase in esterase activity, the exception being the macrophages which contain parasites within their cytoplasm or those with flagellates applied closely to their external surfaces. Only a few specifically stained granules are retained by such phagocytes. Monoamine oxidase activity is limited to fine granules dispersed in the cytoplasm of fibroblasts, epithelial cells, and macrophages of control cultures. Infection of chick liver cultures with T. vaginalis results in lowered enzyme activity in non-phagocytic cells as well as in macrophages with engulfed flagellates and in those whose external surface are invested with the parasites. The number of reactive inclusions appears to increase in trichomonad-free phagocytes of experimental cultures.

Notes: Summary Most of the enzyme activity for urease, dopa oxidase and glucosan phosphorylase is contained in the granules in T. vaginalis. The nucleus lacks reaction in all cases, although a positive reaction in the nuclear membrane and perinuclear concentration of reactive granules is often seen. The failures in the preliminary trials and inconsistency of results obtained with glucosan phosphorylase have been discussed. The similar results obtained with both the techniques used for the demonstration of glucosan phosphorylase chiefly suggest that T. vaginalis contains the “inactive” form of phosphorylase. The cytoplasmic activity for phosphorylase may represent true enzyme site or diffusion of the enzyme or reaction products from the positive granules. This enzyme appears to be labile and some activity is lost during the drying process. The presence of phosphorylase in these organisms along with our previous findings and those of other workers may explain their high glycogen content.

Notes: Summary T. vaginalis contains a high activity for DPN and TPN diaphorases, most of which are localized in cytoplasmic granules which may be mitochondria, and which sometimes show a perinuclear concentration. Occasionally a weak to strong cytoplasmic reaction and some activity in other organelles has been observed, and possible reasons for such reactions have been offered. DPN diaphorase preparations revealed the possibility of cyclic metabolic activity involving nucleo-cytoplasmic relationships, in which the nucleolus appears to play an important role. Aconitase and fumarase could be located only in some cytoplasmic granules of some individuals. The former also appeared along the nuclear membrane and the parabasal body. β-Glucuronidase showed a weak cytoplasmic reaction and a more intense reaction in the small cytoplasmic granules and in or around the nuclear membrane. Transglycosylase was present in very negligible amounts. Supported by FR-00165 from N.T.H.

Notes: Summary 1. Sulphato-cerate solution in the presence of chromium ions and sulphuric acid has been suggested as a new oxidising reagent. 2. The new reagent embodies the oxidising power of both sulphatocerate as well as chromate solutions, it is stable even on prolonged refluxing. 3. It has been shown that glucose, fructose, sucrose, lactose and xylose can be oxidised completely to carbon dioxide and water by the above suggested reagent.

Notes: Summary Oxalic acid is oxidised to carbon dioxide and water with sulphatocerate in dilute sulphuric acid medium. Formic acid is not affected by ceric sulphate under the above conditions. Both oxalic and formic acids are quantitatively oxidised to carbon dioxide and water by the cerate-chromate reagent. Based on this principle a method has been suggested for the estimation of a mixture of oxalic and formic acids.

Notes: Summary In dilute sulphuric acid medium formaldehyde is oxidised to formic acid. Formic acid does not reduce any ceric sulphate under the above conditions. However formic acid is oxidised quantitatively to carbon dioxide and water by cerate-chromate reagent. Based on this principle a method has been worked out for the estimation of a mixture of formaldehyde and formic acid. Moreover it has been shown that the ceratechromate reagent has no effect on acetic acid. Formic acid and acetic acid can be titrated against standard alkali and the total determined as acid. Based on these two different reactions a method has been worked out for the estimation of mixtures of formic and acetic acids.

Notes: Summary 1. The kinetics of the reaction between formic acid and ceric sulphate has been studied in the presence of chromium ions and sulphuric acid and a suitable reaction-mechanism has been proposed. 2. The new reagent embodies the oxidising action of both ceric as well as chromate ions and hence, is a much more powerful oxidant particularly for organic molecules in which the oxidising power of an oxidant is not governed by redox potential only. 3. The new cerate-chromate reagent has the added advantage of stability at higher temperatures.

Notes: Summary 1. A simple volumetric method has been developed for the estimation of mixtures of citric and oxalic acids. 2. The method depends upon the principle that whereas oxalic and citric acids require 2 and 16 equivalents of oxygen for oxidation by pure ceric sulphate in dilute sulphuric acid, they need 2 and 18 equivalents of oxygen respectively when oxidised by ceric sulphate in the presence of chromium ion and concentrated sulphuric acid.