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H C Pitot

Publications and source records attributed to H C Pitot.

At least 271 records · Page 15Linked to original sources
PubMed1982

Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.

Amine Oxidase (Copper-Containing)↗
PubMed1982

DNA damage induced by the antihistaminic drug methapyrilene hydrochloride.

Treatment of primary cultures of rat hepatocytes with the antihistaminic drug, methapyrilene hydrochloride, stimulated DNA-repair synthesis up to 7-fold and caused the formation of alkaline-labile lesions in hepatocellular DNA. These data clearly demonstrate that methapyrilene hydrochloride is a DNA damaging agent. In view of a recent report and our own findings we suggest that this antihistamine has the properties of a complete carcinogen.

Aminopyridines↗
PubMed1982

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.

Animals↗
PubMed1982

Isolation of gamma-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat.

gamma-Glutamyl transpeptidase (gamma GT) positive hepatocytes were isolated from F344 male rats fed 2-acetylaminofluorene. The isolation procedure is rapid and highly selective for cells exhibiting gamma GT on their surface. Suspensions of liver cells obtained from perfusion in situ with a collagenase solution were incubated on Petri dishes coated with affinity purified rabbit anti-gamma GT antibody. gamma GT-positive cells bound to the dish within fifteen minutes and could be recovered as viable cells. Approximately 15% of the gamma GT-positive cells are isolated using this procedure. Novikoff hepatoma cells, a gamma GT-positive cell line, were used to define the parameters of the assay. The binding was both time and temperature dependent. Binding of cells to the anti-gamma GT antibody coated dishes was 50% inhibited by 2.8 mM sodium azide and 86% inhibited by 4.6 mM.

Animals↗
PubMed1982

Hepatic cyst formation in male rats partially hepatectomized as weanlings during short-term feeding of 2-acetylaminofluorene.

Partial hepatectomy (p.h.) of weanling male Sprague-Dawley rats during short-term feeding of 0.03% 2-acetylaminofluorene (AAF) resulted, 12 to 15 months later, in a massive production of hepatic cysts. The weight of the right lateral and caudate lobes plus neoplasms (the left lateral and median lobes were excised at the time of p.h.) accounted for as much as 43% of total body weight. The incidence of hepatic cysts was negligible when animals were not subjected to p.h. during AAF feeding. The feeding of 0.05% phenobarbital (PB) subsequent to AAF + p.h. did not significantly increase the incidence of hepatic cysts. By comparison of the present data with that of other investigators, it can be suggested that susceptibility to cholangioma formation following AAF + p.h. may be determined in part by the developmental stage of the rat and/or its liver at the time of the AAF + p.h. regimen.

2-Acetylaminofluorene↗
PubMed1982

Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism.

Using primary cultures of adult rat hepatocytes, the regulation of the following lipogenic enzymes was studied: glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. The addition to the culture medium of either insulin or triiodothyronine produced a 2-3-fold increase in each of the individual enzyme activities whereas glucagon slightly decreased enzyme activities. The addition to the medium of 8-bromoguanosine 3,'5'-monophosphate had no effect on any of the enzyme activities unless glucose was also added to the culture medium. Glucose addition alone to the culture medium was without any effect; however, glucose enhanced the stimulation of enzyme activity due to insulin. The addition of fructose or glycerol, even in the absence of insulin, increased the activities of each of the enzymes studied 2-3-fold. The increases in enzyme activity brought about by insulin or fructose were apparently the result of de novo enzyme synthesis, as indicated by the observation that the increases were not noted in the presence of cordycepin or cycloheximide. Immunoprecipitation of ATP-citrate lyase from hepatocytes pulse-labeled with [3H]leucine indicated that the induction of this enzyme in response to the addition of fructose or glycerol to the culture medium was the result of an increase in the rate of synthesis of the enzyme. These results indicate that the activity and synthesis of individual enzymes involved in lipogenesis are increased in response to the metabolism of carbohydrate independently in part from hormonal effects.

Animals↗
PubMed1982

Application of quantitative stereology to the evaluation of enzyme-altered foci in rat liver.

The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver. Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number of size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.

Animals↗
PubMed1982

Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses.

The hepatocarcinogenicity of aflatoxin B1 (AFB1) was compared in male Syrian golden hamsters and inbred F344 rats. AFB1 was administered by gavage 5 days/week for 6 weeks at doses of 1 and 2 mg/kg body weight/day to rats and hamsters, respectively; rate did not survive beyond 3 weeks with doses of 2 mg/kg/day. After 6 weeks the animals either received no further treatment or were given 0.1% phenobarbital sodium in the drinking water. ATPase-deficient foci of hepatic parenchymal cells, neoplastic nodules, and hepatocellular carcinomas were observed in liver sections from AFB1-treated rats killed at 6, 14, or 23 weeks; they were not seen in sections from AFB1-treated hamsters killed at 6, 14, or 46 weeks. Each of the 50 AFB1-treated rats developed hepatocellular carcinomas by 46 weeks; many also had cholangiocarcinomas and mixed hepatocellular-cholangiocellular carcinomas. Hepatocellular carcinomas were found in only 2 of 49 AFB1-treated hamsters by 78 weeks. At this time cholangiocarcinomas were found in 15 hamsters, and microscopic cholangiomas were seen in all of the livers. Compared to the rat, the hamster was resistant to the hepatotoxic and hepatocellular carcinogenic effects of AFB1.

Adenoma, Bile Duct↗
PubMed1982

Environmental modifiers in carcinogenesis.

The natural history of neoplastic development can be divided into at least three distinct stages - initiation, promotion and progression. These stages can be defined by biological experimentation and by histopathological characterization of the lesions involved in the various stages. It is now possible, both in human beings and in experimental animals, to distinguish by morphological techniques all stages from very early 'precursor' lesions to the final malignant state. Although the final stage - progression - of the neoplastic process can be altered by therapeutic intervention, the principal site at which the natural history of neoplastic development can be actually and potentially regulated is that of promotion. Promoting agents that are relatively selective for carcinogenesis in several different tissue types have now been identified both in experimental systems and in human beings. In at least two experimental systems - those of epidermal carcinogenesis and hepatocarcinogenesis - the general characteristics of both initiation and promotion are virtually identical. Such studies have demonstrated that the activity of promoting agents exhibits a threshold level as well as a maximal level and that the action of promoting agents is reversible; the identification of promoting agents in our environment is therefore of considerable importance in understanding the nature of and preventing the development of human cancer. In addition, the interpretation of whole-animal bioassays for carcinogenic agents in the environment must take into account the existence of promoting agents and recognize that their action is distinct from that of complete and incomplete carcinogens, in order rationally to extrapolate to situations of human risk.

Carcinoma, Hepatocellular↗
PubMed1982

Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens.

Technical modifications of the quantitative determination of unscheduled DNA synthesis in cultured hepatocytes are described which allow for the rapid identification of potentially carcinogenic chemicals on a large-scale screening basis. The test is based on the biochemical quantification of [methyl-3H]thymidine incorporation into DNA in the presence of hydroxyurea following isolation of nuclei from hepatocytes treated with the agent under study. This procedure ("nuclei procedure") eliminates most of the background radioactivity which otherwise obscures the stimulation of DNA repair synthesis by agents that induce a relatively weak response. By combining the nuclei procedure with a double-labeling technique, test results can be obtained within a few hr after exposure of hepatocytes to the test agents. A test series involving 41 agents confirmed the reliability of the nuclei procedure for the assay of DNA repair synthesis. In addition, chemicals which had yielded conflicting results previously in the autoradiographic hepatocyte DNA repair test, such as 4-acetylaminofluorene, or which had passed unrecognized in previous in vitro tests, such as the potent liver carcinogen methapyrilene hydrochloride, scored clearly positive in our test protocol.

Animals↗
PubMed1981

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.

Animals↗
PubMed1981

Purification and characterization of a new thiol proteinase from rat kidney.

The levels of activity of a new proteinase, termed cathepsin T, in rat tissues were examined. This enzyme had been previously reported to exist in rat liver on the basis of its catalysis of the conversion of multiple forms of tyrosine aminotransferase k(L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5). Kidney was found to be the richest source of cathepsin T activity, exhibiting cathepsin T activity in tissues was kidney much greater than spleen greater than liver greater than small intestine greater than lung. The proteinase activity was not detectable in heart, skeletal muscle, brain and blood. Kidney cathepsin T was purified about 1400-fold to homogeneity with a 20% yield by the purification procedure similar to that used for the liver enzyme (Gohda, E. and Pitot, H.C. (1980) J. Biol. Chem. 255, 7371-7379). Cathepsin T purified from rat kidney was found to be a glycoprotein, the molecular weight of which was between 33,500 and 35,000. Purified kidney cathepsin T converted Form I of tyrosine aminotransferase in the same way as the liver proteinase, with concomitant conversion of 52,500 dalton subunits of the aminotransferase to 48,000 dalton subunits. Kidney cathepsin T showed the same specific activities toward Form I of tyrosine aminotransferase, casein and acid-denatured hemoglobin as did the liver form of the enzyme. Many other characteristics common to the proteinases purified both from rat kidney and liver were found. We have concluded that kidney cathepsin T is the same enzyme as the liver proteinase.

Animals↗
PubMed1981

Role of insulin, glucose, and cyclic GMP in the regulation of glucokinase in cultured hepatocytes.

Following insulin addition to the medium of primary cultures of hepatocytes, the activity of glucokinase increased by 50% during the first 4 h, remained constant for an additional 2 to 3 h, and was followed by a second increase in activity. This biphasic increase in activity was a reflection of the amount of glucokinase enzyme protein present as measured by quantitative immunoprecipitation. The level of cGMP was found to increase sharply 6 h following the addition of insulin and just prior to the second phase of the increase in glucokinase activity. When hepatocytes were cultured in the absence of carbohydrates, only the initial phase of the increase in enzyme activity in response to insulin was observed, and no increase in the level of cGMP was found. However, the addition of 8-bromo-cGMP to the culture medium at 6 h or later could restore the second increase in activity. The addition to the medium of glucose, fructose, pyruvate, or lactate enhanced the insulin-induced increase in glucokinase, but only fructose, pyruvate, and lactate increased the activity of the enzyme in the absence of insulin. When the phosphorylation of hexoses was blocked by the addition of N-acetylglucosamine to the culture medium, an intermediate level of glucokinase activity was observed in response to insulin. The full response was restored by the inclusion of 8-bromo-cGMP in the medium. Both phases of the increase in glucokinase activity were inhibited by cycloheximide, whereas the addition to the culture medium of cordycepin or actinomycin D prevented only the initial increase in activity and not the second increase, which arose from the addition of 8-bromo-cGMP to the cultures.

Acetylglucosamine↗