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Biomedical subjects

S Weinhouse

Publications and source records attributed to S Weinhouse.

At least 19 recordsLinked to original sources
PubMed1986

The role of diet and nutrition in cancer.

Experimental and epidemiologic studies in recent years are pointing to diet as an important contributor to the cancer death toll which in the US this year will reach nearly one half million. Obesity, high fat intake, low fiber content and a dearth of vitamin A- and C-containing fruits and vegetables have been identified as risk factors; but these are not independent variables. The complex network of metabolic mechanisms involved are still obscure and association is not necessarily causation. Experts may agree on the data, but differ on whether we know enough to recommend dietary changes to the public. The Society in its continued efforts toward cancer prevention, has taken the stand that the available evidence, although inferential, is sufficiently solid to share with the public, and its guidelines are compatible with currently acceptable good nutritional practice. This conference should be a landmark of progress in the Society's continuing surveillance of this active field of investigation.

Carcinogens↗
PubMed1983

A microcolorimetric assay of inorganic pyrophosphatase.

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.

Animals↗
PubMed1983

Isozyme alterations, gene regulation and the neoplastic transformation.

Studies of isozyme composition in the rat liver-hepatocellular carcinoma model system have revealed wide-ranging abnormalities of gene expression. Isozymes geared for adult liver function are lost in tumors to varying degrees, depending on growth rate and degree of tumor dedifferentiation; whereas isozymes low or absent in normal adult liver become predominant or sole forms in fast growing, poorly differentiated hepatic tumors. The prevailing pattern is a switch from the adult to fetal forms, thereby indicating that genes coding for adult isozymes are suppressed in liver neoplasms, while genes active in fetal stages but inactivated during normal embryonic development become re-activated in cancer. These isozyme alterations not only are a key to neoplastic behavior, but are a striking example, among many, of pervasive abnormalities of gene expression involving virtually every means of identification of gene products; for example, the expression in tumors of fetal antigens, growth factors, angiogenesis factors, membrane components, and ectopic polypeptide hormones. Most important is the recently discovered activation of normally silent host oncogenes associated with and probably causally related to viral or chemical carcinogenesis. Despite compelling evidence for somatic mutation, these findings argue for abnormal gene regulation in cancer rather than gene mutation. Uriel has proposed that retrodifferentiation to an embryonal stage is a normal response to cell injury, and is an essential step preceding normal re-ordering of gene regulation. Under the influence of a carcinogen, however, normal differentiation may be aborted before differentiation is complete and the resultant transformed cell would then not be subject to normal regulatory restraints. A similar hypothesis has been expressed by Potter in the aphorism "Oncogeny is blocked ontogeny." Evidence exists for a role of 5-methylcytosine as a regulatory site in gene transcription. Jonathan Nyce, in a Ph.D. thesis presented to the Biology Department of Temple University, has proposed a mechanism of chemical carcinogenesis, based on the loss of cytosine residues, and therefore of methylation sites, in DNA as a result either of mispairing of bases resulting from O6-alkylation of guanines in DNA: or by inhibition of cytosine methylation. This plausible proposal provides a molecular basis for the many aberrations of gene regulation in cancer.

Animals↗
PubMed1983

5-Methylcytosine depletion during tumour development: an extension of the miscoding concept.

We propose a general model for neoplastic development which postulates that the loss of methyl groups from 5-methylcytosines (5-mC) involved in the control of gene expression may initiate neoplastic transformation and give rise to the aberrant phenotype of the transformed cell. Interference with normal patterns of methylation can be envisioned to occur by a number of mechanisms: as a result of carcinogen-induced G:C leads to A:T transition leading to a loss of potentially methylatable cytosines; by mutations or chromosome rearrangement which disrupt the integrity of active DNA methylase genes; by separating methylated repressor regions of the genome from the genes they control; by direct interference with DNA methylation, as proposed for ethionine and 5-azacytidine; by spontaneous deamination of 5-mC to thymine, leading to accumulation of 5-mC:G leads to T:A transitions, by virus-induced perturbations in host cell methylation patterns; and by activation of DNA demethylases.

5-Methylcytosine↗
PubMed1983

Subcellular distribution of inorganic pyrophosphatase activity in various normal and neoplastic cell types.

Inorganic pyrophosphatase (PPiase) activity was measured in cell fractions of rat, mouse, and human erythrocytes; normal rat liver; Novikoff hepatoma; Morris 3924A hepatoma; and mouse Ehrlich and Sarcoma 37 ascites tumors. Despite high intracellular activities, when precautions were taken to maintain cell integrity, only negligible activities were found on the surface of intact erythrocytes, Novikoff ascites hepatoma, Ehrlich carcinoma, and Sarcoma 37 cells. Suspensions of intact Ehrlich and Sarcoma 37 cells exhibited low PPiase activity, but this was only about 1 to 2% of the intracellular activity and was completely accounted for by activity present in the suspension medium. It is considered to be due to extrusion of the intracellular enzyme. Systematic fractionation of subcellular components revealed that 92% of the total PPiase activity of rat liver and 97.5% of that of Hepatoma 3924A were in the cytosol. The soluble activity consisted of a major form, accompanied by very low activities of two minor forms, all of which migrate toward the anode on electrophoresis. About 4.5% of the liver activity was present in the mitochondria in two forms, one remaining at the origin and one migrating toward the cathode. The same cytosolic isozymes were present in Hepatoma 3924A, and the cathodic form was present in mitochondria but in much reduced amount. No evidence was obtained for specific isozymes in nuclei or microsomes. Only negligible PPiase activities were found in cell membranes isolated by sucrose gradient centrifugation. These results discount the occurrence of PPiase activity as an ectoenzyme or in the plasma membrane of these cells and point to the cytosol as the major and mitochondria as a minor locus of intracellular PPiase activity.

Animals↗
PubMed1982

Purification and properties of inorganic pyrophosphatase of rat liver and hepatoma 3924A.

Inorganic pyrophosphatase (EC 3.6.1.1) has been purified to electrophoretic homogeneity from the soluble portion of the cytoplasm of rat Hepatoma 3924A and rat liver. It has a specific activity of 600 to 700 mumol inorganic orthophosphate liberated per min per mg protein at 25 degrees, a value in the same range as the highly purified enzymes from yeast and Escherichia coli. By all criteria applied, the hepatoma inorganic pyrophosphatase is identical with the liver enzyme. It is a dimer with subunits with molecular weights of approximately 30,000 to 33,000 and has a pH optimum of 7.4, a Km for pyrophosphate of 5 microM, and a Ka for Mg2+ of 0.3 mM with a pyrophosphate concentration of 0.2 mM. It is not inhibited by high Mg2+ concentrations up to 20 mM. Other metal ions such as Zn2+ and Ca2+ do not activate. Mn2+ activates to less than 10% that of Mg2+ at 0.6 mM and has no effect at 1 mM or higher. In the presence of optimal (4 mM) Mg2+ concentration, Ca2+, Mn2+, Hg2+, and F- at 0.2 mM inhibited strongly, but Zn2+ at 1 mM was not inhibitory. The enzyme had no phosphatase activity toward any of the purine or pyrimidine nucleoside mono-, di-, and triphosphates or toward p-nitrophenyl phosphate, beta-glycerophosphate, glucose 6-phosphate, or glucose 1-phosphate. Bromo- or iodoacetate at high concentration had no inhibitory effect, but p-chloromercuribenzoate and p-chloromercuriphenylsulfonate inhibited strongly at low concentration. The purified enzyme was very unstable but was protected markedly at or above the pH optimum of 7.4 by cysteine, dithiothreitol, and glutathione.

Adenosine Triphosphate↗
PubMed1979

Formation of carbonyl chloride in carbon tetrachloride metabolism by rat liver in vitro.

In order to identify intermediates of CCl4 metabolism, whole, suitably fortified rat liver homogenates were incubated with 14CCl4 in the presence and absence of "pools" of unlabeled suspected intermediates. In the presence of NADH or NADPH, incorporation of radioactivity was rapid and substantial in CO2, lipid, protein, and the acid-soluble fraction. It was not influenced by the presence of large pools of unlabeled chloroform or formate, thus excluding these substances as obligatory intermediates. However, when incubated with L-cysteine, radioactivity incorporation in the acid-soluble fraction was almost doubled, and about one-third of the radioactivity of this fraction was identified as 2-oxothiazolidine 4-carboxylic acid. This substance is formed chemically by condensation of cysteine with carbonyl chloride and has been identified previously by others as a product of chloroform metabolism by liver microsomes in the presence of L-cysteine. Based on current knowledge of CCl4 metabolism, the following aerobic pathway is envisioned: microsomal cleavage to Cl- and .CCl3 and oxidation of the latter to the unstable intermediate, Cl3COH, which loses HCl to yield COCl2. COCl2 is likely to be the major source of CO2 from CCl4 but is probably not the intermediate that binds to lipid and protein. The addition of glutathione had no effect on CCl4 metabolism in rat liver homogenate, suggesting that glutathione S-transferases, which catalyze other dehalogenation reactions, do not play a role in CCl4 metabolism.

Aerobiosis↗
PubMed1977

Metabolism of [3H]-methylcholanthrene in the perfused rat liver.

The metabolism of [3H]-3-methylcholanthrene (3-MC) was studied in the isolated, perfused rat liver. Following addition of 250 mug to the perfusion fluid, 3-MC disappeared rapidly. After 2 hr, approximately 34% of the radioactivity was excreted in the bile, 6% remained in the perfusate, and 60% was found in the liver. Of the liver radioactivity, 80% was unchanged 3-MC, 11% was conjugated metabolites, 4% was free hydroxymetabolites, and 4% was nonextractable, presumably bound to macromolecules. Of the perfusate radioactivity, 82% was conjugated metabolites, 3% was free hydroxymetabolites, and 15% was unchanged 3-MG. A similar distribution was observed in intact, bile-cannulated rats, but biliary excretion was about one-fourth as high with double the i.v.-injected dose. Biliary excretion in perfused livers rose rapidly during the first 30 to 40 min, then decreased steadily. It was nearly twice as high in male as in female rat livers. Pretreatment of rats with 3-MC more than doubled the biliary excretion rate over the first 20 to 30 min in livers of both sexes and raised that of the female to that of the male rat liver. Neither retinol acetate nor 7,8-benzoflavone had any appreciable effect on biliary excretion of 3-MC metabolites. 2-Diethylaminoethyl-2,2-diphenylvalerate, a well-known microsomal oxygenase inhibitor, lowered excretion by 80 to 90% and lengthened the lag period, and dibutyryl-3',5'-cyclic adenosine monophosphate markedly increased the rate of excretion of 3-MC metabolites. Fractionation of bile by chromatography on Sephadex LH-20 revealed six well-defined peaks of radioactivity. In contrast, bile of intact rats given 3-MC gave a pattern on Sephadex chromatography consisting of only three peaks. Preliminary data suggest that these consist of conjugates of dihydroxymetabolites as well as more highly hydroxylate derivatives. The data obtained indicate that the perfused liver is an appropriate experimental model for studies on the hepatobiliary metabolism of carcinogens.

Animals↗
PubMed1977

Urea synthesis in Novikoff and Morris hepatomas.

The rates of urea synthesis in rat liver and in a series of rat liver neoplasms with widely different growth rates and degree of differentiation were investigated using tissue slices incubated in a Krebs-Ringer bicarbonate buffer. Urea synthesis did not occur in fast-growing, poorly differentiated Novikoff and Morris 3924A hepatomas, but it did occur in slow-growing, well- and highly differentiated hepatomas; however, there was no correlation with growth rate or degree of differentiation. Urea synthesis was comparable with normal liver, at about 32 mumoles/hr/g tissue, in the slow-growing Morris hepatomas 21, 28A, 47C, and 44; but it was very low in two other slow-growing, highly differentiated hepatomas, 9618A and 20. The well-differentiated Morris hepatoma 5123C had intermediate levels of urea synthesis. This pattern of urea synthesis closely paralleled the previously reported activity of carbamyl phosphate synthetase in these tumors. The rate of urea synthesis was normal in livers of Buffalo rats bearing fast- or slow-growing hepatomas in low urea synthesis rates, but it was markedly lowered in the livers of rats bearing large, slow-growing tumors with high urea synthesis rates. Urea synthesis in liver declined as the tumors increased in size. The total rate of urea synthesis in liver and tumor, as well as the concentrations of urea in the serum and urine of tumor-bearing animals, remained remarkably constant throughout the period of tumor growth, suggesting the existence of a homeostatic mechanism that controls the urea cycle activity in accordance with the synthetic activity of the tumor. In parabiotic animals, carbamyl phosphate synthetase activity and urea synthesis were lowered in the host livers of partners bearing tumors with high carbamyl phosphate synthetase- and urea-synthetic activity, but there was no significant effect on urea cycle activity in the normal partners. This result discounts the likelihood of a circulating humoral factor that controls hepatic urea cycle activity.

Animals↗