Sheldon M. Epstein

Lysosomal Stability during Phagocytosis of Aspergillus flavus Spores by Alveolar Macrophages of Cortisone-Treated Mice

Control mice and those treated with cortisone were exposed to aerosols of viable spores of Aspergillus flavus. Fifteen to 20 minutes later, animals were killed, and alveolar macrophages were obtained by tracheobronchial lavage. Electron-microscopic examination of these cells revealed that, whereas the lysosomes of control macrophages showed extensive attraction and fusion with the phagocytic membranes surrounding spores, the lysosomes of macrophages from animals treated with cortisone revealed little, if any, interaction. This diminished lysosomal response in forming phagocytic vacuoles may be important in the subsequent development of hyphal bronchopneumonia which frequently, occurs in cortisonetreated mice exposed to spores of A. flavus.

Increased cyclic AMP levels in malignant hepatic nodules of ethionine treated rats

Abstract Cyclic AMP levels were measured in vivo and in vitro in uninvolved liver, benign and malignant hepatic nodules of ethionine treated rats. The in vivo cyclic AMP concentration of 5.0 ± 0.5 nmoles/gm protein in malignant nodules was twice that in benign nodules and uninvolved liver. Values in tissue slices after sacrifice of anesthetized animals were identical to the in vivo levels. Cyclic AMP levels were higher in animals sacrificed by decapitation, but were not influenced by 20 minutes incubation. Concentrations in malignant nodules still exceeded those in the other two tissues. The increased cyclic AMP concentrations in malignant nodules persisted whether the data was expressed on the basis of wet weight, protein or DNA.

Growth in vitro of Cells from Hyperplastic Nodules of Liver Induced by 2-Fluorenylacetamide or Aflatoxin B1

Cell suspensions obtained from hyperplastic nodules induced in rat liver by either of the two hepatic carcinogens, 2-fluorenylacetamide or aflatoxin B1, show growth when cultured in vitro. No growth of cells from liver adjacent to the hyperplastic nodules or from liver of control rats has been obtained so far under comparable conditions. Hepatocarcinoma cells induced by 2-fluorenylacetamide grow readily in vitro but behave differently. These findings suggest that some nonmalignant cells capable of growth in vitro arise during liver carcinogenesis prior to the appearance of unequivocal cancer. Cultures of such cells may offer new avenues for the study of liver carcinogenesis.

Altered and distorted DNA from a premalignant liver lesion induced by 2-fluorenylacetamide

Abstract DNA isolated from a precancerous population of liver cells, weeks after the termination of the dietary exposure to the carcinogen 2-fluorenylacetamide (2-FAA) was found to show obvious alterations in its chemical and physical properties. The DNA had (I) an altered absorption spectrum in the range 350–310 mμ, (2) a small population with an increased buoyant density as observed in equilibrium centrifugation in CsCl, and (3) denatured and distorted fibres as observed with electron microscopy, when compared with DNA from normal control liver, the surrounding non-hyperplastic liver or regenerating liver following partial hepatectomy. These findings indicate the possibility that an observable alteration in DNA may play a role in carcinogenesis and suggest a possible mechanism for the progressive nature of the cellular changes during cancer induction.

Pyridine nucleotides in the thyroid: III. A method for determination of oxidized and reduced diphosphopyridine nucleotides using 6-phospho-[I14C]-gluconate

Abstract Methods are presented for assay of micromicromolar quantities of oxidized and reduced NAD following synthesis to NADP utilizing excess ATP and NAD kinase (ATP:NAD 2′-phosphotransferase, EC 2.7.1.23). NADP + is assayed by minor modifications of prior methodology in which the yield of 14 CO 2 is proportional to NADP + when excess 6-phospho[I 14 C]gluconate and excess 6-phosphogluconate dehydrogenase (6-phospho- d -gluconate:NADP + oxidoreductase, EC 1.1.1.44) are present. The reduced nucleotides are assayed in their oxidized state following reaction with excess α-ketoglutarate, NH 4 + and glutamate dehydrogenase ( l -glutamate:NAD + (NADP + ) oxidoreductase (deaminating), EC 1.4.1.3). The prior methodology for NADP + assay has been modified, without any sacrifice to sensitivity, so that incubations can be performed in reduced volumes and at room temperature. These modifications have enabled the assay procedures to be performed in center wells, temporarily sealed in counting vials, thereby eliminating the time consuming step of hyamine transfer. Using these methods equivalent amounts of nucleotides per gram wet weight tissue were obtained with different amount of tissue extract. Recovery of internal standards was virtually complete. Values for a number of mammalian tissues, obtained by utilization of the present methodology, are presented and are similar to those previously reported. The influence of extraction procedure upon nucleotide concentration obtained in tissues is mentioned.

Pyridine nucleotides in the thyroid: VI. DPN kinase activity of thyroid homogenate obtained from slices incubated with and without thyroid stimulating hormone

Abstract Thyroid slices incubated with and without TSH have been homogenized and assayed for DPN kinase (ATP:DPN 2′-phosphotransferase, EC 2.7.1.23) activity. Such activity was increased when homogenates from TSH incubated slices were incubated with ATP and DPN and [1- 14 C]-6-phosphogluconate oxidation measured as an indication of TPN formation. In similar experiments when TPN was measured directly, homogenates obtained from TSH incubated slices also had greater TPN levels than homogenates prepared from control slices. However, in these experiments since net synthesis of TPN was not observed, the results could also be explained by decreased rate of destruction as well as by increased synthesis of TPN. Subcellular fractionation of thyroid slices localized most of the DPN kinase to the nuclear fraction while the increased TPN of TSH incubated slices was in the supernatant. The TSH stimulation of glucose oxidation was not inhibited by a concentration of actinomycin D which caused a 90% reduction in [8- 14 C]adenine incorporation into nucleic acids. Vasopressin, which has been reported to directly stimulate thyroxine release from the thyroid, did not stimulate glucose oxidation in thyroid slices. TSH did not influence glucose oxidation of gastric mucosa and submandibular gland, two tissues which also share with thyroid the ability to concentration iodide but are not TSH responsive in terms of this parameter.